Variant LovD polypeptides and their uses

ABSTRACT

The present disclosure provides acyltransferases useful for synthesizing therapeutically important statin compound.

1. CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a Continuation of U.S. patent applicationSer. No. 15/899,907, filed Feb. 20, 2018, now U.S. Pat. No. 10,590,445,which is a Divisional application of U.S. patent application Ser. No.15/372,788, filed Dec. 8, 2016, now U.S. Pat. No. 9,932,616, which is aContinuation application of U.S. patent application Ser. No. 14/710,893,filed May 13, 2015, now U.S. Pat. No. 9,546,388, which is a Divisionalof U.S. patent application Ser. No. 14/465,467, filed Aug. 21, 2014, nowU.S. Pat. No. 9,181,570, which is a Divisional of U.S. patentapplication Ser. No. 13/755,113, filed on Jan. 31, 2013, now U.S. Pat.No. 8,846,362, which claims the benefit under 35 U.S.C. § 120 to U.S.patent application Ser. No. 12/890,134, filed Sep. 24, 2010, now U.S.Pat. No. 8,383,382, and under 35 U.S.C. § 119(e) to U.S. Prov. Pat.Appln. Ser. Nos. 61/247,253 and 61/247,274, both filed Sep. 30, 2009,the contents of all of which are incorporated herein in their entiretiesby reference thereto.

The official copy of the Sequence Listing is submitted concurrently withthe specification as an ASCII formatted text file via EFS-Web, with afile name of “CX2_022WO_SL_230412-REV.txt”, a creation date of Apr. 27,2012, and a size of 5,521 bytes. This Sequence Listing text file isidentical to the Sequence Listing text file submitted in the parentapplication Ser. No. 12/890,134 on Apr. 27, 2012, which was a correctedcopy of the original Sequence Listing text file “CX2-022.txt” that wasoriginally filed with the parent application Ser. No. 12/890,134 on Sep.24, 2010. The Sequence Listing filed via EFS-Web is part of thespecification and is incorporated in its entirety by reference herein.

2. BACKGROUND

Simvastatin is a semi-synthetic analog of the natural fungal polyketidelovastatin which can be isolated from the fermentation broth ofAspergillus terreus. Simvastatin and lovastatin are both marketed byMerck Co. as cholesterol-lowering drugs that reduce the risk of heartdisease: simvastatin as ZOCOR® and lovastatin as MEVACOR®.

Lovastatin (illustrated in FIG. 1) is a potent inhibitor ofhydroxymethylglutaryl coenzyme A reductase, the rate-limiting enzyme inthe cholesterol biosynthetic pathway (Xie et al., 2006, Chemistry &Biology 13:1161-1169). The analog simvastatin (also illustrated inFIG. 1) is more effective in treating hypercholesterolemia (Manzoni &Rollini, 2002, Appl. Microbiol. Biotechnol. 58:555-564; Istan &Diesenhofer, 2001, Science 292:1160-1164). Substitution of theα-methylbutyrate side chain of lovastatin with the α-dimethylbutyrateside chain found in simvastatin significantly increases its inhibitoryproperties while lowering undesirable side effects (Klotz, Ulrich, 2003,Arzneimittel-Forschung 53: 605-611).

Because of the clinical importance of simvastatin, various multi-stepsyntheses starting from lovastatin have been described (see, e.g., WO2005/066150; US Application No. 2005/0080275; US Application No.2004/0068123; U.S. Pat. No. 6,833,461; WO 2005/040107; Hoffman et al.,1986, J. Med. Chem. 29:849-852; Schimmel et al., 1997, Appl. Environ.Microbiol. 63:1307-1311).

The gene cluster for lovastatin biosynthesis has been previouslydescribed (see, e.g., U.S. Pat. No. 6,391,583; Kennedy et al., 1999,Science 284:1368-1372; Hutchinson et al., 2000, Antonie Van Leeuwenhoek78:287-295). Encoded in this gene cluster is the 46 kD enzyme LovD,which catalyzes the last step of lovastatin biosynthesis.

Briefly, the decalin core and HMG-CoA moieties that mimic portions ofthe lovastatin compound are synthesized in vivo by lovastatin nonaketidesynthase (LNKS) and three accessory enzymes. The 2-methylbutyrate sidechain of lovastatin is synthesized in vivo by lovastatin diketidesynthase (LDKS) and covalently attached to the acyl carrier domain ofLovF via a thioester linkage. LovD, an acyltransferase, is then able toselectively transfer the 2-methylbutyrate group from LDKS to the C8hydroxyl group of monacolin J in a single step to yield lovastatin (Xieet al., 2006, Chemistry & Biology 13:1161-1169).

It has recently been discovered that the LovD acyltransferase has broadsubstrate specificity towards the acyl carrier, the acyl substrate andthe decalin acyl acceptor (Xie et al., 2006, Chem. Biol. 13:1161-1169).For example, LovD can efficiently catalyze acyl transfer from CoAthioesters or N-acetylcysteamine (“SNAC”) thioesters to monacolin J(id.). Significantly, when α-dimethylbutyryl-SNAC was used as the acyldonor, LovD was able to convert monacolin J and 6-hydroxy-6-desmethylmonacolin J into simvastatin and huvastatin, respectively (id.). Usingan E. coli strain engineered to overexpress LovD as a whole-cellbiocatalyst, preparative quantities of simvastatin were synthesized in asingle fermentation step (id.).

The above studies demonstrate that LovD acyltransferase is an attractiveenzyme for the biosynthesis of pharmaceutically importantcholesterol-lowering drugs such as simvastatin. However, in subsequentexperiments carried out with isolated LovD enzyme, stability andreaction rate proved problematic (Xie & Tang, 2007 Appl. Environ.Microbiol. 73:2054-2060). Specifically, it was found that LovDprecipitates readily (hours) at high protein concentrations (˜100 μM)and slowly (days) at lower concentrations (˜10 μM) (id.). In addition,it was found that the very desired product, simvastatin, competes forthe LovD enzyme, significantly impeding the overall net rate ofacylation (id.).

The LovD enzyme is also highly prone to mis-folding and aggregates whenover-expressed in E. coli, making even whole-cell biocatalysis systemsless than ideal for commercial production (Xie et al., 2009, Biotech.Bio Eng. 102:20-28).

In an effort to increase LovD solubility without loss of catalyticactivity, mutants of wild-type A. terreus LovD have been studied.Replacing the cysteine residues at positions 40 and 60 (Cys40 and Cys60)with alanine residues yielded improvements in both enzyme solubility andwhole-cell biocatalytic activity (id.). Further mutagenesis experimentsconverting these two residues to small or polar amino acids showed thatCys40→Ala (“C40A”) and Cys60→Asn (“C60N”) mutations are the mostbeneficial, yielding 27% and 26% increases, respectively, in whole-cellbiocatalytic activity (id.). When combined, these mutations provedadditive, with the C40A/C60N double mutant exhibiting approximately 50%increases in both solubility and whole-cell biocatalytic activity.

Despite their improved properties, these LovD mutants are unsuitable forlarge scale production of simvastatin in cell-free systems. Additionalvariants or mutants of wild-type A. terreus LovD enzymes that exhibitimproved properties as compared to the wild-type and/or known mutantswould be desirable.

3. SUMMARY

As discussed above, the LovD gene of A. terreus encodes an acyltransferase (hereinafter called “LovD polypeptide,” “LovD enzyme,” “LovDacyltransferase” or “LovD”) capable of converting monacolin J, thehydrolysis product of the natural product lovastatin, to simvastatin.The inventors of the present disclosure have discovered that LovDpolypeptides including mutations at certain residue positions exhibitimproved properties as compared to the wild-type LovD polypeptideproduced by A. terreus (SEQ ID NO:2).

Accordingly, in one aspect, the present disclosure provides variant LovDpolypeptides that have one or more improved properties as compared towild-type LovD polypeptide from A. terreus (SEQ ID NO:2). Generally, theLovD variants include one or more mutations at selected positions thatcorrelate with one or more improved properties, such as increasedcatalytic activity, increased thermal stability, reduced aggregationand/or increased stability to cell lysis conditions. The variant LovDpolypeptides can include one or more mutations from a single category(for example, one or more mutations that increase catalytic activity),or mutations from two or more different categories. By selectingmutations correlating with specific properties, variant LovDpolypeptides suitable for use under specified conditions can be readilyobtained.

Positions in the wild-type LovD polypeptide sequence of SEQ ID NO:2 atwhich mutations have been found that correlate with one or more improvedproperties, such as increased catalytic activity include, but are notlimited to, A123, M157, 5164, 5172, L174, A178, N191, L192, A247, R250,S256, A261, G275, Q297, L361, V370 and N391. Additional positions atwhich mutations have been found which correlate with one or moreimproved properties, such as thermal stability, include, but are notlimited to, Q241, A261, Q295 and Q412. Yet further positions at whichmutations have been found that correlate with one or more improvedproperties, such as reduced aggregation, include but are not limited to,N43, D96 and H404. Positions at which mutations were found thatcorrelated with one or more improved properties, such as increasedstability included, but are not limited to, C40, C60 and D254.

Positions in the wild-type LovD polypeptide sequence of SEQ ID NO:2 atwhich mutations having no detrimental effect (or at which mutationsimproved the properties of the LovD enzyme) were found include, but arenot limited to, 14, A9, K26, R28, 135, C40, S41, N43, C60, 5109, 5142,A184V, N191S, A261, L292, Q297, L335, A377, A383, N391 and H404.Particular embodiments with improved properties can include, but are notlimited to, LovD polypeptides with mutations at positions L174 and A178,and optionally from zero to about 30 additional mutations. In a specificembodiment, the additional mutations are at positions selected from thepositions identified above. In some embodiments, LovD polypeptides withimproved properties include mutations at positions A123, L174, A178,N191, A247 and L361, and from zero up to about 26 additional mutations.In a specific embodiment, the additional mutations are at positionsselected from the positions identified above.

Specific, exemplary mutations of the wild-type LovD polypeptide of SEQID NO:2 that correlate with increased catalytic activity include, butare not limited to, A123P, M157V, S164G, S172N, L174F, A178L, N191G,L192I, A247S, R250K, S256T, A261H, G275S, Q297G, L361M, V370I and N391S.

Specific, exemplary mutations of the wild-type LovD polypeptide of SEQID NO:2 that correlate with increased thermal stability include, but arenot limited to, Q241M, A261H, Q295R and Q412R.

Specific, exemplary mutations of the wild-type LovD polypeptide of SEQID NO:2 that correlate with reduced aggregation include, but are notlimited to, N43R, D96R and H404K.

Specific exemplary mutations of the wild-type LovD polypeptide of SEQ IDNO:2 that correlate with increased stability include, but are notlimited to, C40R, C60R and D254E.

In addition to the specific, exemplary mutations disclosed above, it hasbeen discovered that LovD polypeptides can tolerate a range ofadditional specific mutations at other positions without detrimentaleffect. Indeed, in some instances, the additional mutations also conferthe LovD polypeptides with beneficial or improved properties. Theseadditional mutations include, but are not limited to, the followingspecific and exemplary mutations: I4N, A9V, K26E, R28K, R28S, I35L,C40A, C40V, C40F, S41R, N43Y, C60F, C60Y, C60N, C60H, S109C, S142N,A184T, A184V, N191S, A261T, A261E, A261V, L292R, Q297E, L335M, A377V,A383V, N391D and H404R. Variant LovD polypeptides may include one ormore mutations at these additional positions.

In some embodiments, the variant LovD polypeptides described hereininclude the following two mutations: L174F and A178L, and optionallyfrom zero to about 30 additional mutations. In a specific embodiment,the optional additional mutations are selected from the variousmutations identified above.

In some embodiments, the variant LovD polypeptides described herein willinclude at least the following mutations: A123P, L174F, A178L, N191(S orG), A247S and L361M, and from zero up to about 26 additional mutationsselected from the various different mutations identified above.

In general, variant LovD polypeptides including greater numbers ofmutations exhibit greater catalytic activity. For example, whereas aspecific variant including mutations at two residue positions (L174F andA178L) exhibited approximately two-fold greater activity than wild-typeLovD, and a specific variant including mutations at six residuepositions (Variant 120 in Table 1) exhibited approximately 12-foldgreater catalytic activity than wild-type LovD, one specific variantincluding 28 mutations (Variant 114 in Table 1), one specific variantincluding 29 mutations (Variant 116 in Table 1) and one specific variantincluding 32 mutations (Variant 118 in Table 1) each exhibitedapproximately 1550-fold greater catalytic activity than wild-type LovD.Table 1 discloses numerous variant LovD polypeptides exhibiting fromabout 10- to about 1550-fold greater activity than the wild-type LovDpolypeptide of SEQ ID NO:2. Using these exemplary specific variant LovDpolypeptides, additional LovD variant polypeptides having greater thanabout 10-, 20-, 30-, 40-, 50-, 60-, 70-, 80-, 90-, 100-, 150-, 200-,300-, 400-, 500-, 600-, 700-, 800-, 900-, 1000-, 1100-, 1200-, 1300-,1400-, 1500-, 1600-, 1700-fold, or even greater, activity than thewild-type LovD polypeptide of SEQ ID NO:2 can be readily obtained.

The specific variant LovD polypeptides of Table 1 can also be used toreadily obtain variant LovD polypeptides that have specifiedcombinations of improved properties.

The various variant LovD polypeptides described herein may alsooptionally include one or more conservative mutations in addition to themutations described above. Typically, such optional conservativemutations will not comprise more than about 20%, 15%, 10%, 8%, 5%, 3%,2%, or 1% of the overall sequence. Without intending to be bound by anyparticular theory of operation, it is believed that the amino acid atposition 76 may be involved in catalysis. Mutations at this residueposition should be preferably avoided. It is also believed that theamino acids at position 79, 148, 188 and/or 363 may contribute tocatalysis. In some embodiments, variant LovD polypeptides that includeoptional conservative mutations will contain from 1 to 20 suchmutations. In additional embodiments, variant LovD polypeptides includetruncated polypeptides wherein 1 to 15 amino acids may be omitted fromthe N-terminus and 1 to 6 amino acids may be omitted from theC-terminus.

In another aspect, the disclosure provides polynucleotides encoding thevariant LovD polypeptides described herein. In some embodiments theencoding polynucleotides are part of an expression vector comprising oneor more control sequences suitable for directing expression of theencoding sequence in a host cell. In some embodiments, the expressionvector is suitable for expressing variant LovD polypeptides in abacterium, such as but not limited, to an E. coli, and includes apromoter sequence, such as a lac promoter sequence, operably linked tothe encoding sequence. In such polynucleotides the codons of theencoding sequence can be optimized for expression in a particular hostcell of interest.

In yet another aspect, the present disclosure provides host cellscomprising a variant LovD encoding polynucleotide or expression vector.In some embodiments, the host cell is a bacterium such as but notlimited to E. coli. The host cells can be used to produce crude orpurified preparations of specific variant LovD polypeptides, or,alternatively, they can be used as whole-cell preparations in themethods described herein to produce simvastatin and huvastatin.

The variant LovD polypeptides described herein esterify the C8 hydroxylgroup of monacolin J in the presence of an α-dimethylbutyryl thioesterco-substrate to yield simvastatin. Accordingly, in another aspect, thepresent disclosure provides methods of making simvastatin utilizing thevariant LovD polypeptides described herein. The methods generallycomprise contacting monacolin J with a variant LovD polypeptide in thepresence of an α-dimethylbutyryl thioester co-substrate under conditionswhich yield simvastatin. In some embodiments, analogues of monacolin Jmay be used as precursor substrates to the formation of other statins,such as, but not limited to, huvastatin.

The variant LovD polypeptides described herein recognize a number ofdifferent α-dimethylbutyryl thioester co-substrates, including by way ofexample and not limitation, α-dimethylbutyryl-S—N-acetylcysteamine(“DMB-S-NAC”), α-dimethylbutyryl-S-methylthioglycolate (“DMB-S-MTG”),α-dimethylbutyryl-S-methyl mercaptopropionate “DMB-S-MMP”),α-dimethylbutyryl-S-ethyl mercaptoproprionate (“DMB-S-EMP”),α-dimethylbutyryl-S-methyl mercaptobutyrate (“DMB-S-MMB”), andα-dimethylbutyryl-S-merceaptopropionic acid (“DMB-S-MPA”). Any of thesethioester co-substrates, or mixtures of such thioester co-substrates,can be used in the methods described herein.

In some embodiments, the reaction is carried out in vitro with anisolated variant LovD polypeptide, which can be purified or unpurifiedprior to use. In some embodiments, enzyme tags may be added to eitherterminus of the LovD polypeptide in order to enable binding to a solidcarrier. In some specific embodiments, the variant LovD polypeptide isisolated and purified prior to use. In other specific embodiments, thevariant LovD polypeptide is supplied as a crude lysate of host cellsengineered to express the variant LovD polypeptide, with or withoutremoval of the cell debris from the cell lysate.

The monacolin J substrate can be included in the reaction mixture inpurified form, or, alternatively, it can be generated in situ byhydrolysis of lovastatin. Accordingly, in some embodiments, the reactionis carried out in a single pot as a two-step process starting fromlovastatin.

The methods can be carried out under a variety of conditions, dependingupon, among other factors, the activity of the specific variant LovDpolypeptide being used. A typical reaction includes about 1 to 250 g/L,also 25 to 200 g/L, and often 1 to 200 g/L, monacolin J substrate oranalogue thereof, excess thioester co-substrate (for example, from about1.0 to 10.0 equiv, also 1.0 to 5.0 equiv, and often 1.0 to 4.0 equiv)and about 0.1 to 10 g/L LovD variant polypeptide. In some embodiments, atypical reaction may include 20 to 200 g/L, 50 to 200 g/L, or 50 to 150g/L monacolin J substrate or analogue thereof. In some embodiments, atypical reaction may include from 1.0 to 2.0, from 1.0 to 1.5, or from1.1 to 1.3 equivalents of thioester co-substrate. In some embodiments,0.2 to 10 g/L, 0.5 to 10 g/L, 0.5 to 5 g/L, 0.75 to 2.5 g/L, or 0.75 to1.5 g/L of LovD variant polypeptide may be used. The pH of the reactionmixture, the temperature at which the reaction is carried out and theduration of the reaction will depend upon, among other factors, thespecific LovD variant polypeptide being used. Most reactions can becarried out at a pH in the range of pH 7.5 to pH 10.5, also pH 8.0 to pH10.0, and often pH 8 to pH 9.5, and a temperature in the range of about20 to 50° C., also 20 to 30° C., and often 20 to 40° C., forapproximately 2 to 54 hrs, 5 to 48 hrs or 10 to 48 hrs. Reactionscarried out with variant LovD polypeptides including mutations thatcorrelate with increased thermal stability can be carried out at highertemperatures, typically in a range of about 30 to 40° C., depending uponthe particular variant being used.

The thiol by-product of the acyl transfer reaction may inhibit LovDpolypeptides. Accordingly, it may be desirable to include in thereaction mixture one or more scavenging agents that might improvereaction rate and/or yield by removing or scavenging these thiolby-products. Scavenging includes, without limitation, chemicalmodification of thiol by-products, such as by oxidation. Suitablescavenging agents may include, but are not limited to, compounds thatreact with the thiol by-product and agents capable of chelating,adsorbing, absorbing or removing the thiol by-product. In someembodiments where a scavenging agent is used, the scavenging agent isactivated charcoal. When used, the activated charcoal can be included inthe reaction mixture in quantities ranging from 1 to 30 g/L, 2 to 20 g/Land 5 to 15 g/L.

The monacolin J substrate may be in the form of a salt, such as anammonium or sodium salt. Reaction of the monacolin J substrate, such asthe sodium or ammonium salts of monacolin J, may optionally be run inthe presence of a scavenger, such as activated charcoal. Monacolin J mayalso be in the form of an ammonium salt. When the ammonium salt ofmonacolin J is converted to the ammonium salt of simvastatin, theammonium salt of simvastatin may be precipitated from the reactionmedium, for example, from water.

Other aspects and advantages of the disclosure will be apparent from thedetailed description that follows.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides a diagram illustrating a pathway for synthesizingsimvastatin from lovastatin or monacolin J;

FIG. 2 provides a polynucleotide sequence encoding wild type LovD enzymefrom Aspergillus terreus that has been codon-optimized for expression inE. coli. (SEQ ID NO:1); and

FIG. 3 provides the polypeptide sequence encoded by the sequence of FIG.2 (SEQ ID NO:2).

5. DETAILED DESCRIPTION

In certain aspects, the present disclosure provides LovD variantpolypeptides that are capable of transferring an acyl group from certainthioester co-substrates to monacolin J and analogs or derivativesthereof to yield therapeutically important statin compounds, such as butnot limited to simvastatin and huvastatin. The LovD variants haveimproved properties as compared to the wild-type LovD acyltransferaseobtainable from A. terreus (SEQ ID NO:2), and can be used in cell-basedor cell-free systems to efficiently and cost-effectively produce statinssuch as simvastatin from readily available starting materials, such aslovastatin and monacolin J.

5.1. Abbreviations

For the purposes of the descriptions herein, the abbreviations used forthe genetically encoded amino acids are conventional and are as follows:

Amino Acid Three-Letter Abbreviation One-Letter Abbreviation Alanine AlaA Arginine Arg R Asparagine Asn N Aspartate Asp D Cysteine Cys CGlutamate Glu E Glutamine Gln Q Glycine Gly G Histidine His H IsoleucineIle I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe FProline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine TyrY Valine Val V

When peptide or polypeptide sequences are presented as a string ofone-letter or three-letter abbreviations (or mixtures thereof), thesequences are presented in the N→C direction in accordance with commonconvention.

5.2. Definitions

The technical and scientific terms used in the descriptions herein willhave the meanings commonly understood by one of ordinary skill in theart, unless specifically defined otherwise. Accordingly, the followingterms are intended to have the following meanings.

“Wild-type LovD,” “Wild-type LovD enzyme,” “Wild-type LovD polypeptide,”and/or “Wild-type LovD acyltransferase” refers to the acyltransferaseenzyme obtainable from A. terreus encoded by the LovD gene and having anamino acid sequence corresponding to SEQ ID NO:2. This enzyme can use athioester to regiospecifically acylate the C8 hydroxyl group ofmonacolin J or 6-hydroxy-6-des-methyl monacolin J so as to producesimvastatin or huvastatin. See, e.g., Xie et al., 2006, “Biosynthesis ofLovastatin Analogs with a Broadly Specific Acyltransferase,” Chem. Biol.13:1161-1169.

“Coding sequence” refers to that portion of a nucleic acid orpolynucleotide (e.g., a gene, mRNA, cDNA, etc.) that encodes a peptideor polypeptide.

“Naturally occurring” or “wild-type” refers to the form of a material orsubstance as found in nature. For example, a naturally occurring orwild-type polypeptide or polynucleotide sequence is a sequence presentin an organism that can be isolated from a source in nature and whichhas not been modified by human manipulation.

“Recombinant” when used with reference to, e.g., a cell, a nucleic acid,or a polypeptide, refers to a material, or a material corresponding tothe natural or native form of the material, that has been modified in amanner such that it exists in nature, or is identical to a material thatexists in nature, but is produced or derived from synthetic materialsand/or by natural materials that have been manipulated in some way.Non-limiting examples include, among others, cells engineered to expressnucleic acid sequences that are not found within the native(non-recombinant) forms of the cell, or that express native genes foundwithin the non-recombinant form of the cell at levels that differ fromtheir native expression levels.

“Percentage of sequence identity,” “percent identity,” and/or “percentidentical” are used herein to refer to comparisons betweenpolynucleotide sequences or polypeptide sequences, and are determined bycomparing two optimally aligned sequences over a comparison window,wherein the portion of the polynucleotide or polypeptide sequence in thecomparison window may comprise additions or deletions (i.e., gaps) ascompared to the reference sequence in order to effect optimal alignment.The percentage identity is calculated by dividing the number of matchedportions in the comparison window by the total number of positions inthe comparison window, and multiplying by 100. The number of matchedpositions in the comparison window is the sum of the number of positionsof the comparison polynucleotide or polypeptide in the window that areidentical in sequence to the reference polynucleotide or polypeptide andthe number of positions of the reference polynucleotide or polypeptidein the comparison window that align with a gap in the comparisonpolynucleotide or polypeptide. Determination of optimal alignment andpercent sequence identity is performed using the BLAST and BLAST 2.0algorithms (see, e.g., Altschul et al., 1990, J. Mol. Biol. 215:403-410and Altschul et al., 1997, Nucleic Acids Res. 25(17):3389-3402).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information website.

Briefly, the BLAST analyses involve first identifying high scoringsequence pairs (HSPs) by identifying short words of length W in thequery sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold (Altschul et al, 1990, supra). These initial neighborhood wordhits act as seeds for initiating searches to find longer HSPs containingthem. The word hits are then extended in both directions along eachsequence for as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). For aminoacid sequences, a scoring matrix is used to calculate the cumulativescore. Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, M=5, N=−4, and a comparison of bothstrands. For amino acid sequences, the BLASTP program uses as defaults awordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoringmatrix (see Henikoff & Henikoff, 1989, Proc. Nat'l Acad. Sci. USA89:10915). Numerous other algorithms are available that functionsimilarly to BLAST in providing percentage identity between sequences.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, 1981, Adv. Appl. Math.2:482, by the homology alignment algorithm of Needleman & Wunsch, 1970,J. Mol. Biol. 48:443, by the search for similarity method of Pearson &Lipman, 1988, Proc. Nat'l Acad. Sci. USA 85:2444, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe GCG Wisconsin Software Package), or by visual inspection (seegenerally, Current Protocols in Molecular Biology, F. M. Ausubel et al.,eds., Current Protocols, a joint venture between Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc., 1995 Supplement).

Additionally, determination of sequence alignment and percent sequenceidentity can employ the BESTFIT or GAP programs in the GCG WisconsinSoftware package (Accelrys, Madison Wis.), using default parametersprovided.

“Reference sequence” refers to a specified sequence to which anothersequence is compared. A reference sequence may be a subset of a largersequence, for example, a segment of a full-length gene or polypeptidesequence. Generally, a reference sequence is at least 20 nucleotide oramino acid residues in length, at least 25 residues in length, at least50 residues in length, or the full length of the nucleic acid orpolypeptide. Since two polynucleotides or polypeptides may each (1)comprise a sequence (i.e., a portion of the complete sequence) that issimilar between the two sequences, and (2) may further comprise asequence that is divergent between the two sequences, sequencecomparisons between two (or more) polynucleotides or polypeptide aretypically performed by comparing sequences of the two polynucleotidesover a comparison window to identify and compare local regions ofsequence similarity.

The term “reference sequence” is not intended to be limited to wild-typesequences, and can include engineered, variant and/or altered sequences.

“Comparison window” refers to a conceptual segment of at least about 20contiguous nucleotide positions or amino acids residues wherein asequence may be compared to a reference sequence of at least 20contiguous nucleotides or amino acids and wherein the portion of thesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. The comparison window can be longer than 20contiguous residues, and includes, optionally 30, 40, 50, 100, or longerwindows.

“Substantial identity” refers to a polynucleotide or polypeptidesequence that has at least about 80% sequence identity with a referencesequence. In many embodiments, sequences that share “substantialidentity” will be at least 85%, 90%, 95%, 96%, 97%, 98%, or even 99%identical to the reference sequence.

The phrase “corresponding to”, “reference to” or “relative to” when usedin the context of the numbering of a given amino acid sequence orpolynucleotide sequence refers to the numbering of the residues of aspecified reference sequence when the given amino acid sequence orpolynucleotide sequence is compared to the specific reference sequence.In other words, the residue number or residue position of a givenpolymer is designated with respect to the reference sequence rather thanby the actual numerical position of that residue within the given aminoacid or polynucleotide sequence. For example, a given amino acidsequence, such as that of a variant LovD polypeptide, can be aligned toa reference sequence by introducing gaps to optimize residue matchesbetween the two sequences. In these cases, although the gaps arepresent, the numbering of the residue in the given amino acid orpolynucleotide sequence is made with respect to the reference sequenceto which it has been aligned.

“Increased catalytic activity,” when used in the context of the variantLovD polypeptide described herein, refers to a LovD polypeptide thatexhibits increased conversion of substrate (for example monacolin J or asalt thereof) to product (for example simvastatin or a salt thereof) ascompared to a reference LovD polypeptide, as measured in an in vitro orin vivo assay. The origin of the increase in catalytic activity is notcritical. Thus, the increase could be due to changes in one or more ofK_(m), V_(max) or K_(cat) or due to decreased substrate inhibition. Insome embodiments, for purposes of comparison, catalytic activity can beconveniently expressed in terms of the percentage of substrate convertedto product per unit of enzyme in a specified period of time. Enzymesthat convert a greater percentage of substrate to product per unit perperiod of time than a reference enzyme assayed under identicalconditions have increased catalytic activity as compared to thereferenced enzyme.

Methods and assays for measuring the catalytic activity of enzymes arewell-known in the art. Specific examples useful for LovD polypeptidesare provided in the Examples section. For comparative assays carried outwith crude cell lysates, identical host cells and expression systemsshould be used. In addition, the number of cells and amount of LovDpolypeptide in each preparation should be determined, as is known in theart.

“Thermostable” or “thermal stable” in the context of LovD polypeptidesrefer to variant LovD polypeptides that retain at least about 50% oftheir catalytic activity when exposed to a temperature of 30° C. for aperiod of 3 hrs. as compared to the catalytic activity exhibited by thatvariant LovD polypeptide at 25° C. under the same reaction conditions.

“Residue” refers to an amino acid when used in the context ofpolypeptides and a nucleotide when used in the context ofpolynucleotides.

“Hydrophilic amino acid or residue” refers to an amino acid having aside chain exhibiting a hydrophobicity of less than zero according tothe normalized consensus hydrophobicity scale of Eisenberg et al., 1984,J. Mol. Biol. 179:125-142. Genetically encoded hydrophilic amino acidsinclude Thr (T), Ser (S), His (H), Glu (E), Asn (N), Gln (Q), Asp (D),Lys (K) and Arg (R).

“Acidic amino acid or residue” refers to a hydrophilic amino acid havinga side chain exhibiting a pKa value of less than about 6 when the aminoacid is included in a peptide or polypeptide. Acidic amino acidstypically have negatively charged side chains at physiological pH due toloss of a hydrogen ion. Genetically encoded acidic amino acids includeGlu (E) and Asp (D).

“Basic amino acid or residue” refers to a hydrophilic amino acid orresidue having a side chain exhibiting a pKa value of greater than about6 when the amino acid is included in a peptide or polypeptide. Basicamino acids typically have positively charged side chains atphysiological pH due to association with hydronium ion. Geneticallyencoded basic amino acids include Arg (R), His (H) and Lys (K).

“Polar amino acid or residue” refers to a hydrophilic amino acid havinga side chain that is uncharged at physiological pH, but which has atleast one bond in which the pair of electrons shared in common by twoatoms is held more closely by one of the atoms. Genetically encodedpolar amino acids include Asn (N), Gln (Q), Ser (S) and Thr (T).

“Hydrophobic amino acid or residue” refers to an amino acid having aside chain exhibiting a hydrophobicity of greater than zero according tothe normalized consensus hydrophobicity scale of Eisenberg et al., 1984,J. Mol. Biol. 179:125-142. Genetically encoded hydrophobic amino acidsinclude Pro (P), Ile (I), Phe (F), Val (V), Leu (L), Trp (W), Met (M),Ala (A) and Tyr (Y).

“Aromatic amino acid or residue” refers to a hydrophilic or hydrophobicamino acid having a side chain that includes at least one aromatic orheteroaromatic ring. Genetically encoded aromatic amino acids includePhe (F), Tyr (Y) and Trp (W). Owing to the pKa of its heteroaromaticring nitrogen, His (H) is classified herein as a hydrophobic or basicresidue. It is additionally classified as an aromatic residue due to itsheteroaromatic side chain.

“Constrained amino acid or residue” refers to an amino acid that has aconstrained geometry. Herein, constrained residues include Pro (P).

“Non-polar amino acid or residue” refers to a hydrophobic amino acidhaving a side chain that is uncharged at physiological pH and which hasbonds in which the pair of electrons shared in common by two atoms isgenerally held equally by each of the two atoms (i.e., the side chain isnot polar). Genetically encoded non-polar amino acids include Gly (G),Leu (L), Val (V), Ile (I), Met (M) and Ala (A).

“Aliphatic amino acid or residue” refers to a hydrophobic amino acid orresidue having an aliphatic hydrocarbon side chain. Genetically encodedaliphatic amino acids include Ala (A), Val (V), Leu (L) and Ile (I).

Cysteine is unusual in that it can form disulfide bridges with other Cysresidues or other sulfanyl- or sulfhydryl-containing amino acids.“Cysteine-like residues” include cysteine and other amino acids thatcontain sulfhydryl moieties that are available for formation ofdisulfide bridges. The ability of Cys (and other amino acids with —SHcontaining side chains) to exist in a polypeptide in either the reducedfree —SH or oxidized disulfide-bridged form affects whether itcontributes net hydrophobic or hydrophilic character to the polypeptide.While Cys exhibits a hydrophobicity of 0.29 according to the normalizedconsensus scale of Eisenberg (Eisenberg et al., 1984, supra), it is tobe understood that for purposes of the present disclosure, Cys iscategorized into its own unique group.

“Small amino acid or residue” refers to an amino acid having a sidechain that is composed of a total of three or fewer carbon and/orheteroatoms (excluding the α-carbon and hydrogens). The small aminoacids or residues may be further categorized as aliphatic, non-polar,polar or acidic small amino acids or residues, in accordance with theabove definitions. Genetically-encoded small amino acids include Ala(A), Val (V), Cys (C), Asn (N), Ser (S), Thr (T) and Asp (D).

“Hydroxyl-containing amino acid or residue” refers to an amino acidcontaining a hydroxyl (—OH) moiety. Genetically-encodedhydroxyl-containing amino acids include Ser (S) Thr (T) and Tyr (Y).

“Conservative” amino acid substitutions or mutations refer to thosesubstitutions and mutations in which amino acid residues of a referencepolypeptide are replaced with amino acid residues having similarphysicochemical properties. Substitutions and mutations that areconsidered conservative are well-known in the art. In some embodiments,conservative substitutions and mutations are those in which an aminoacid of a particular class is replaced with another amino acid withinthat same class (for example, aliphatic aliphatic). Exemplaryconservative substitutions are provided below:

Residue Possible Conservative Mutations A, L, V, I Other aliphatic (A,L, V, I) Other non-polar (A, L, V, I, G, M) G, M Other non-polar (A, L,V, I, G, M) D, E Other acidic (D, E) K, R Other basic (K, R, H) N, Q, S,T Other polar Y, W, F Other aromatic (Y, W, F, H) C, P None

“Isolated” refers to a substance that has been removed from the sourcein which it naturally occurs. A substance need not be purified in orderto be isolated. For example, a variant LovD polypeptide recombinantlyproduced in a host cell is considered isolated when it is removed orreleased from the cell. A variant LovD polypeptide contained within acrude cell lysate fraction is considered “isolated” for purposes of thepresent disclosure.

“Purified” refers to a substance that has been rendered at leastpartially free of contaminants and other materials that typicallyaccompany it. Substances can be purified to varying degrees. A substanceis “substantially pure” when a preparation or composition of thesubstance contains less than about 1% contaminants. A substance is“essentially pure” when a preparation or composition of the substancecontains less than about 5% contaminants A substance is “pure” when apreparation or composition of the substance contains less than about 2%contaminants. For substances that are “purified to homogeneity,”contaminants cannot be detected with conventional analytical methods.

5.3. LovD Variant Polypeptides

As discussed in the Background Section, the polypeptide encoded by thelovD gene of A. terreus is an acyltransferase that catalyzes the laststep of lovastatin biosynthesis. This LovD acyltransferase has broadsubstrate specificity towards the acyl carrier, the acyl substrate andthe decalin acceptor (see, e.g., Xie et al., 2006, Chem. Biol.12:1161-1169) such that the enzyme can be used to transfer acyl groupsfrom thioester co-substrates to decalins such as monacolin J and6-hydroxy-6-desmethyl monacolin J to yield therapeutically importantstatin compounds. An exemplary reaction is illustrated in FIG. 1 (boxedregion). As illustrated in this FIG. 1 LovD acyltransferasecatalytically transfers the α-dimethylbutyryl group of thioester (14) tomonacolin J (12) to yield simvastatin (16), making it an attractivetarget for the preparation of this pharmaceutically important statin.

Despite its attractive catalytic properties, attempts to use isolated A.terreus LovD acyltransferase and certain mutants thereof in vitro forproduction of simvastatin have not been very successful. Stability,solubility, mis-folding, aggregation and reaction rate all provedproblematic (see, e.g., Xie & Tang, 2007, Appln. Environ. Microbiol.73:2054-2060; Xie et al., 2009, Biotech. Bio. Eng. 102:20-28).

The instant disclosure provides variant LovD polypeptides that, like thewild-type LovD acyltransferase from A. terreus (SEQ ID NO:2),catalytically transfer an acyl group from a thioester co-substrate tomonacolin J (or its analogues or derivatives) to yield simvastatin.These variants include mutations at specified positions and exhibit oneor more improved properties as compared to the wild-type LovD andtransferase of SEQ ID NO:2.

Skilled artisans will appreciate that lovastatin, monacolin J andsimvastatin, as well as their analogues and derivatives can exist invarious forms including acid, ester, amide and lactone forms. The acid,ester, amide and lactone forms can also be in the form of salts. Theacid (R═—OH), ester (R═—O (alkyl)), amide (R═—N(alkyl)₂) and lactoneforms of these compounds are illustrated below. Unless stated otherwise,“lovastatin” as used herein includes the acid, ester, amide, lactone andsalt forms, “monacolin J” as used herein includes the acid, ester,amide, lactone and salt forms and “simvastatin” as used herein includesthe acid, ester, amide, lactone and salt forms. These forms can be usedin the methods described herein.

Mutation experiments carried out with wild-type A. terreus LovDacyltransferase revealed that mutations at specified positions correlatewith improvements in catalytic activity, thermal stability, stabilityunder conditions of cell lysis and aggregation properties. It was alsodiscovered that a range of mutations could be incorporated at certainpositions that, while not providing an identifiable improved property,did not deleteriously affect the overall properties of the polypeptide.All of these various mutations, as well as additional optionalconservative mutations, can be used alone and/or in combinations toyield LovD variant polypeptides having specified properties.Significantly, unlike the wild-type A. terreus LovD acyltransferase, thevariant LovD polypeptides described herein can be isolated and used inin vitro reaction systems to produce therapeutically important statincompounds.

Mutations of the wild-type A. terreus LovD acyltransferase of SEQ IDNO:2 that have been found to correlate with increased catalytic activityinclude, but are not limited to, A123P, M157V, S164V, S172N, L174F,A178L, N191G, L192I, A247S, R250K, S256T, A261H, G275S, Q297G, L361M,V370I and N391S.

Mutations of the wild-type A. terreus LovD acyltransferase of SEQ IDNO:2 that have been found to correlate with increased thermal stabilityinclude, but are not limited to, Q241M, A261H, Q295R and Q412R.

Mutations of the wild-type A. terreus LovD acyltransferase of SEQ IDNO:2 that have been found to correlate with reduced aggregation include,but are not limited to, N43R, D96R and H404K.

Mutations of the wild-type A. terreus LovD acyltransferase of SEQ IDNO:2 that have been found to correlate with increased enzyme stabilityunder conditions of cell lysis include, but are not limited to, C40R,C60R and D245E.

Positions within the wild-type A. terreus LovD acyltransferase of SEQ IDNO:2 that can be mutated without deleterious effect include, but are notlimited to, I4N, A9V, K26E, R28K, R28S, I35L, C40A, C40V, C40F, S41R,N43Y, C60F, C60Y, C60N, C60H, S109C, S142N, A184T, A184V, N191S, A261T,A261E, A261V, L292R, Q297E, L355M, A377V, A383V, N391D and H404R.

One important class of variant LovD polypeptides includes LovDpolypeptides that exhibit increased catalytic activity as compared tothe wild-type A. terreus LovD acyltransferase of SEQ ID NO:2. It hasbeen discovered that including greater numbers of mutations increasesthe catalytic activity of the LovD polypeptide. As illustrated by theexemplary embodiments of variant LovD polypeptides provided in Table 1,infra, combinations of mutations from those identified above can beselected to obtain variant LovD polypeptides having specified catalyticand other properties. In Table 1, the indicated mutations are relativeto SEQ ID NO:2 and the Relative Activity is relative to the activity ofthe wild-type LovD acyltransferase from A. terreus. Conditions used forthe activity assay are provided in the Examples section.

TABLE 1 Relative Variant No. Mutations Activity 120 A123P; L174F; A178L;N191S; A247S; L361M; + 4 I35L; A123P; L174F; A178L; N191S; A247S;L361M; + 6 A123P; L174F; A178L; N191S; A247S; G275S; L361M; + 8 A123P;L174F; A178L; N191S; A247S; R250K; L361M; + 10 A123P; L174F; A178L;N191S; A247S; Q297E; L361M; + 12 R28K; A123P; L174F; A178L; N191S;A247S; L361M; + 14 A123P; L174F; A178L; A184T; N191S; A247S; L361M; + 16A123P; L174F; A178L; N191S; A247S; Q297E; L361M; + 18 A123P; L174F;A178L; N191S; L192I; A247S; L361M; + 20 A123P; L174F; A178L; N191S;A247S; R250K; L361M; + 22 A123P; L174F; A178L; N191S; A247S; A261E;L361M; + 24 A123P; L174F; A178L; N191S; A247S; L361M; H404R; + 26 K26E;A123P; L174F; A178L; N191S; A247S; L361M; + 28 A123P; S172N; L174F;A178L; N191S; A247S; G275S; L361M; ++ 30 A123P; M157V; S172N; L174F;A178L; N191S; A247S; G275S; L361M; ++ 32 A123P; L174F; A178L; N191G;A247S; G275S; L361M; + 34 A123P; L174F; A178L; N191S; A247S; G275S;L335M; L361M; + 36 A123P; L174F; A178L; N191S; A247S; G275S; L361M;H404K; + 38 A123P; L174F; A178L; A184V; N191S; A247S; G275S; L361M; + 40D96R; A123P; L174F; A178L; N191S; A247S; G275S; L361M; + 42 A123P;L174F; A178L; N191G; A247S; G275S; L361M; + 44 A123P; L174F; A178L;N191S; A247S; G275S; L335M; L361M; + 46 A123P; L174F; A178L; N191S;A247S; G275S; L292R; L361M; + 48 A123P; L174F; A178L; N191S; L192I;A247S; R250K; G275S; Q297E; L361M; ++ 50 A123P; L174F; A178L; N191S;L192I; A247S; R250K; G275S; L361M; ++ 52 K26E; C40R; N43Y; A123P; L174F;A178L; N191S; L192I; A247S; G275S; ++ L361M; 54 K26E; C40R; A123P;L174F; A178L; N191S; L192I; A247S; G275S; L361M; ++ 56 K26E; A123P;L174F; A178L; N191S; A247S; G275S; L361M; + 58 A9V; K26E; A123P; M157V;S172N; L174F; A178L; N191S; L192I; A247S; +++ R250K; G275S; Q297E;L361M; A383V; 60 K26E; A123P; M157V; S172N; L174F; A178L; N191S; L192I;A247S; R250K; +++ G275S; L361M; 62 A123P; M157V; S172N; L174F; A178L;N191G; A247S; G275S; L335M; ++ L361M; 64 N43R; D96R; A123P; M157V;S172N; L174F; A178L; N191S; A247S; G275S; ++ L361M; H404K; 66 A9V; K26E;A123P; M157V; S172N; L174F; A178L; N191S; L192I; A247S; +++ R250K;S256T; G275S; Q297E; L361M; A383V; 68 A9V; K26E; S41R; A123P; M157V;S172N; L174F; A178L; N191S; L192I; +++ A247S; R250K; A261V; G275S;Q297E; L361M; A383V; 70 A9V; K26E; R28K; A123P; M157V; S164G; S172N;L174F; A178L; N191G; +++ L192I; Q241M; A247S; R250K; G275S; Q297E;L361M; V370I; A383V; 72 A9V; K26E; R28K; C40R; A123P; M157V; S164G;S172N; L174F; A178L; +++ N191G; L192I; Q241M; A247S; R250K; G275S;Q297E; L361M; V370I; A383V; 74 A9V; K26E; R28K; C40R; A123P; M157V;S164G; S172N; L174F; A178L; +++ N191G; L192I; Q241M; A247S; R250K;G275S; Q297E; L361M; V370I; A383V; 76 A9V; K26E; A123P; M157V; S164G;S172N; L174F; A178L; N191G; L192I; +++ A247S; R250K; G275S; Q297E;L361M; V370I; A377V; A383V; 78 A9V; K26E; N43R; A123P; M157V; S164G;S172N; L174F; A178L; N191G; ++++ L192I; Q241M; A247S; R250K; G275S;Q297E; L361M; V370I; A383V; H404K; 80 A9V; K26E; N43R; D96R; A123P;M157V; S164G; S172N; L174F; A178L; ++++ N191G; L192I; Q241M; A247S;R250K; G275S; Q297E; L361M; V370I; A383V; H404K; 82 A9V; K26E; D96R;A123P; M157V; S164G; S172N; L174F; A178L; N191G; ++++ L192I; Q241M;A247S; R250K; G275S; Q297E; L361M; V370I; A383V; 84 A9V; K26E; D96R;A123P; M157V; S172N; L174F; A178L; N191G; L192I; +++ Q241M; A247S;R250K; G275S; Q297E; L361M; V370I; A383V; 86 A9V; K26E; N43R; D96R;A123P; M157V; S164G; S172N; L174F; A178L; +++ N191G; L192I; A247S;R250K; G275S; Q297E; L361M; V370I; A383V; H404K; 88 A9V; K26E; N43R;D96R; A123P; M157V; S164G; S172N; L174F; A178L; ++++ N191G; L192I;Q241M; A247S; R250K; G275S; Q297E; L361M; V370I; A383V; 90 A9V; K26E;R28S; N43R; A123P; M157V; S164G; S172N; L174F; A178L; ++++ N191G; L192I;Q241M; A247S; R250K; D254E; G275S; Q297E; L361M; V370I; A383V; H404K; 92A9V; K26E; N43R; A123P; M157V; S164G; S172N; L174F; A178L; N191G; ++++L192I; Q241M; A247S; R250K; A261V; G275S; Q295R; Q297E; L361M; V370I;A383V; H404K; Q412R; 94 A9V; K26E; N43R; A123P; M157V; S164G; S172N;L174F; A178L; N191G; ++++ L192I; Q241M; A247S; R250K; A261V; G275S;Q297E; L361M; V370I; A383V; H404K; 96 A9V; K26E; N43R; A123P; M157V;S164G; S172N; L174F; A178L; N191G; ++++ L192I; Q241M; A247S; R250K;A261V; G275S; Q295R; Q297E; L361M; V370I; A383V; N391D; H404K; 98 A9V;K26E; N43R; A123P; M157V; S164G; S172N; L174F; A178L; N191G; ++++ L192I;Q241M; A247S; R250K; S256T; A261V; G275S; Q297G; L361M; V370I; A383V;N391S; H404K; 100 A9V; K26E; N43R; A123P; M157V; S164G; S172N; L174F;A178L; N191G; ++++ L192I; Q241M; A247S; R250K; S256T; A261V; G275S;Q297G; L361M; V370I; A383V; N391S; H404K; 102 A9V; K26E; N43R; S109C;A123P; M157V; S164G; S172N; L174F; A178L; ++++ N191G; L192I; Q241M;A247S; R250K; S256T; A261V; G275S; Q297G; L361M; V370I; A383V; N391S;H404K; 104 A9V; K26E; N43R; A123P; M157V; S164G; S172N; L174F; A178L;N191G; ++++ L192I; Q241M; A247S; R250K; S256T; A261H; G275S; Q297G;L361M; V370I; A383V; N391S; H404K; 106 A9V; K26E; N43R; A123P; M157V;S164G; S172N; L174F; A178L; N191G; ++++ L192I; Q241M; A247S; R250K;S256T; A261H; G275S; Q295R; Q297G; L361M; V370I; A383V; N391S; H404K;Q412R; 108 I4N; A9V; K26E; R28S; N43R; A123P; M157V; S164G; S172N;L174F; A178L; ++++ N191G; L192I; Q241M; A247S; R250K; S256T; A261H;G275S; Q297G; L361M; V370I; A383V; N391S; H404K; 110 I4N; A9V; K26E;R28S; N43R; A123P; M157V; S164G; S172N; L174F; A178L; ++++ N191G; L192I;Q241M; A247S; R250K; D254E; S256T; A261H; G275S; Q297G; L361M; V370I;A383V; N391S; H404K; 112 I4N; A9V; K26E; R28S; N43R; S109C; A123P;M157V; S164G; S172N; L174F; ++++ A178L; N191G; L192I; Q241M; A247S;R250K; S256T; A261H; G275S; Q295R; Q297G; L361M; V370I; A383V; N391S;H404K; Q412R; 114 I4N; A9V; K26E; R28S; I35L; N43R; D96R; A123P; M157V;S164G; S172N; ++++ L174F; A178L; N191G; L192I; Q241M; A247S; R250K;S256T; A261H; G275S; Q297G; L335M; L361M; V370I; A383V; N391S; H404K;116 I4N; A9V; K26E; R28S; I35L; N43R; D96R; S109C; A123P; M157V; S164G;++++ S172N; L174F; A178L; N191G; L192I; Q241M; A247S; R250K; S256T;A261H; G275S; Q297G; L335M; L361M; V370I; A383V; N391S; H404K; 118 I4N;A9V; K26E; R28S; I35L; C40R; N43R; C60R; D96R; S109C; A123P; ++++ M157V;S164G; S172N; L174F; A178L; N191G; L192I; Q241M; A247S; R250K; D254E;S256T; A261H; G275S; Q297G; L335M; L361M; V370I; A383V; N391S; H404K;

In Table 1, variants with a relative activity of “+” exhibited fromabout 10 to 50-fold greater activity than wild-type; variants with arelative activity of “++” exhibited from about 50 to 100-fold greateractivity than wild type; variants with a relative activity of “+++”exhibited from about 100 to 500-fold greater activity than wild type;and variants with a relative activity of “++++” exhibited from about 500to 2000-fold greater activity than wild type.

In some embodiments, mutations are selected from those identified aboveto yield variant LovD polypeptides exhibiting at least two-fold greatercatalytic activity than the wild-type A. terreus LovD. Such variant LovDpolypeptides have amino acid sequences that correspond to SEQ ID NO:2,but include at least the following two mutations: L174F and A178L, andoptionally from one to about 30 additional mutations selected from thevarious mutations identified above, and optionally from about one to 20additional conservative mutations.

In some embodiments, mutations are selected from those identified aboveto yield variant LovD polypeptides exhibiting at least about 10-foldgreater catalytic activity than the wild-type A. terreus LovD. Suchvariant LovD polypeptides have amino acid sequences that correspond toSEQ ID NO:2, but include at least the following mutations: A123P, L174F,A178F, N191(S or G), A247S and L361M, and from zero to about 26additional mutations selected from the various mutations identifiedabove, and optionally from about 1 to about 20 additional conservativemutations. Specific exemplary variant LovD polypeptides are provided inTable 1.

In some embodiments, the variant LovD polypeptides have amino acidsequences that correspond to the variant LovD polypeptides of Table 1and include one or more conservative amino acid substitutions, typicallyat residue positions that are not mutated as compared to SEQ ID NO:2. Insome embodiments, the number of conservative amino acid substitutions isselected such that the sequence of a specific variant LovD polypeptideretains at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with thespecific reference variant LovD polypeptide.

The mutations identified above, along with the specific exemplaryvariant LovD polypeptides provided in Table 1, can be used to createvariant LovD polypeptides having specific properties. For example,variant LovD polypeptides having greater thermal stability than thewild-type A. terreus acyltransferase of SEQ ID NO:2 can be obtained byincluding one of or more mutations identified above that correlate withincreased thermal stability. By “mixing and matching” mutations from thedifferent categories, variant LovD polypeptides having improvements inone or more different properties can be readily obtained.

In some embodiments, mutations are selected such that the variant LovDpolypeptides are thermally stable.

In some embodiments, mutations are selected such that the variant LovDpolypeptides are more stable to conditions of cell lysis than thewild-type A. terreus acyltransferase of SEQ ID NO:2. Increased stabilityto cell lysis can be measured by pre-incubating the lysate at anelevated temperature (for example, 35 to 45° C.) and finding residualactivity.

In some embodiments, mutations are selected such that the variant LovDpolypeptides exhibit less aggregation than the wild-type A. terreusacyltransferase of SEQ ID NO:2 as determined in, for example, 100 mMtriethanolamine buffer at a pH of 8 to 9 and at a temperature of 25° C.

Skilled artisans will appreciate that in many instances, the full lengthvariant LovD polypeptide is not necessary for the enzyme to retaincatalytic activity. Accordingly, truncated analogs and catalyticallyactive fragments of the variant LovD polypeptides are contemplated. Forexample, in some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 aminoacids may be omitted. In additional embodiments, variant LovDpolypeptides include truncated polypeptides wherein 1 to 15 amino acidsmay be omitted from the N-terminus and 1 to 6 amino acids may be omittedfrom the C-terminus. Any specific truncated analog or fragment can beassessed for catalytic activity utilizing the assays provided in theExamples section.

Likewise, additional amino acid residues can be added to one or bothtermini without deleteriously affecting catalytic activity. Accordingly,while many exemplary embodiments of the variant LovD polypeptidesdescribed herein contain 413 amino acid residues, analogs that includefrom about 1 to about 434 additional amino acids at one or both terminiare also contemplated. The additional sequence may be functional ornon-functional. For example, the additional sequence may be designed toaid purification, act as a label, or perform some other function. Thus,the variant LovD polypeptides of the disclosure can be in the form offusion polypeptides in which the variant LovD polypeptides (or fragmentsthereof) are fused to other polypeptides, such as, by way of example andnot limitation, antibody tags (e.g., myc epitope), purificationssequences (e.g., His tags for binding to metals), and cell localizationsignals (e.g., secretion signals).

The variant LovD polypeptides can be obtained by conventional means,including chemical synthesis and recombinant expression. Polynucleotidesand host cells useful for recombinant expression are described below.Variant LovD polypeptides obtained by synthetic means can includenon-genetically encoded amino acids, as is known in the art. Commonlyencountered non-encoded amino acids that can be included in syntheticvariant LovD polypeptides include, but are not limited to:2,3-diaminopropionic acid (Dpr); α-aminoisobutyric acid (Aib);ε-aminohexanoic acid (Aha); β-aminovaleric acid (Ava); N-methylglycineor sarcosine (MeGly or Sar); ornithine (Orn); citrulline (Cit);t-butylalanine (Bua); t-butylglycine (Bug); N-methylisoleucine (MeIle);phenylglycine (Phg); cyclohexylalanine (Cha); norleucine (Nle);naphthylalanine (Nal); 2-chlorophenylalanine (Oct);3-chlorophenylalanine (Mcf); 4-chlorophenylalanine (Pcf);2-fluorophenylalanine (Off); 3-fluorophenylalanine (Mff);4-fluorophenylalanine (Pff); 2-bromophenylalanine (Obf);3-bromophenylalanine (Mbf); 4-bromophenylalanine (Pbf);2-methylphenylalanine (Omf); 3-methylphenylalanine (Mmf);4-methylphenylalanine (Pmf); 2-nitrophenylalanine (Ont);3-nitrophenylalanine (Mnf); 4-nitrophenylalanine (Pnf);2-cyanophenylalanine (Oct); 3-cyanophenylalanine (Mcf);4-cyanophenylalanine (Pcf); 2-trifluoromethylphenylalanine (Ott);3-trifluoromethylphenylalanine (Mtf); 4-trifluoromethylphenylalanine(Ptf); 4-aminophenylalanine (Paf); 4-iodophenylalanine (Pit);4-aminomethylphenylalanine (Pamf); 2,4-dichlorophenylalanine (Opef);3,4-dichlorophenylalanine (Mpcf); 2,4-difluorophenylalanine (Opff);3,4-difluorophenylalanine (Mpff); pyrid-2-ylalanine (2pAla);pyrid-3-ylalanine (3pAla); pyrid-4-ylalanine (4pAla); naphth-1-ylalanine(1nAla); naphth-2-ylalanine (2nAla); thiazolylalanine (taAla);benzothienylalanine (bAla); thienylalanine (tAla); furylalanine (fAla);homophenylalanine (hPhe); homotyrosine (hTyr); homotryptophan (hTrp);pentafluorophenylalanine (5ff); styrylkalanine (sAla); authrylalanine(aAla); 3,3-diphenylalanine (Dfa); 3-amino-5-phenypentanoic acid (Afp);penicillamine (Pen); 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid(Tic); β-2-thienylalanine (Thi); methionine sulfoxide (Mso);N(w)-nitroarginine (nArg); homolysine (hLys);phosphonomethylphenylalanine (pmPhe); phosphoserine (pSer);phosphothreonine (pThr); homoaspartic acid (hAsp); homoglutamic acid(hGlu); 1-aminocyclopent-(2 or 3)-ene-4 carboxylic acid; pipecolic acid(PA), azetidine-3-carboxylic acid (ACA);1-aminocyclopentane-3-carboxylic acid; allylglycine (aOly);propargylglycine (pgGly); homoalanine (hAla); norvaline (nVal);homoleucine (hLeu), homovaline (hVal); homoisoleucine (hIle);homoarginine (hArg); N-acetyl lysine (AcLys); 2,4-diaminobutyric acid(Dbu); 2,3-diaminobutyric acid (Dab); N-methylvaline (MeVal);homocysteine (hCys); homoserine (hSer); hydroxyproline (Hyp) andhomoproline (hPro). Additional non-encoded amino acids of which thepolypeptides described herein may be comprised will be apparent to thoseof skill in the art (see, e.g., the various amino acids provided inFasman, 1989, CRC Practical Handbook of Biochemistry and MolecularBiology, CRC Press, Boca Raton, Fla., at pp. 3-70 and the referencescited therein, all of which are incorporated by reference). These aminoacids may be in either the L- or D-configuration and are preferable inthe L-configuration.

When utilized, such non-encoded amino acids are generally selected suchthat they are conservative substitutions as compared to the referencesequence. Non-encoded amino acids can impart the variant LovDpolypeptides with improved properties, such as, for example, increasedsolubility in desired solvents, increased stability to proteases, etc.Such non-encoded amino acids will typically be included at only a fewresidue positions, for example, such that greater than 98% or 99% of thevariant LovD polypeptide is composed of genetically encoded amino acids.

5.4. Nucleic Acids

In another aspect, the present disclosure provides polynucleotidesencoding the variant LovD polypeptides. The polynucleotides may beoperatively linked to one or more regulatory sequences that control geneexpression to create a recombinant polynucleotide capable of expressingthe variant LovD polypeptide. Expression constructs comprising apolynucleotide sequence encoding a variant LovD polypeptide can beintroduced into appropriate host cells to express the correspondingvariant LovD polypeptide.

Because of the known genetic code, availability of a polypeptidesequence provides a description of all the polynucleotides capable ofencoding that polypeptide. The degeneracy of the genetic code yields anextremely large number of nucleic acids encoding a specific variant LovDpolypeptide. Thus, having identified a particular polypeptide sequence,those skilled in the art could make any number of different nucleicacids encoding that polypeptide sequence by simply modifying thesequence of one or more codons in a way that does not alter the encodedsequence. In this regard, the present disclosure specificallycontemplates each and every possible individual polynucleotide thatencodes a specified polypeptide sequence, and all such individualnucleic acids are to be considered specifically disclosed for anyvariant LovD polypeptide disclosed herein.

In some embodiments, the polynucleotides comprise codons that areoptimized for expression in a specific type of host cell. Codon usageand biases for a variety of different types of microorganisms are wellknown, as are optimized codons for expression of specific amino acids ineach of these microorganisms. (See, e.g., Andersson, S G, and C GKurland, 1990, Microbiol. Mol. Biol. Rev. 54(2): 198-210. Ermolaeva, MD., 2001, Current Issues in Molecular Biology 3(4): 91-97).

In some embodiments, the polynucleotides encoding the variant LovDpolypeptides can be provided as expression vectors, where one or morecontrol sequences are present to regulate the expression of thepolynucleotides. Manipulation of the isolated polynucleotide prior toits insertion into a vector may be desirable or necessary depending onthe expression vector. Techniques for modifying polynucleotides andnucleic acid sequences utilizing recombinant DNA methods are well knownin the art. Guidance is provided in Sambrook et al., 2001, MolecularCloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor LaboratoryPress; and Current Protocols in Molecular Biology, Ausubel. F. ed.,Greene Pub. Associates, 1998, updates to 2006.

In some embodiments, the control sequences include among others,promoters, leader sequences, polyadenylation sequences, propeptidesequences, signal peptide sequences, and transcription terminators. Forbacterial host cells, suitable promoters for directing transcriptionencoding sequence include, but are not limited to, promoters obtainedfrom the E. coli lac operon, E. coli trp operon, bacteriophage □□,Streptomyces coelicolor agarase gene (dagA), Bacillus subtilislevansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM),Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacilluslicheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylBgenes, and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978,Proc. Natl Acad. Sci. USA 75: 3727-3731), as well as the tac promoter(DeBoer et al., 1983, Proc. Natl Acad. Sci. USA 80: 21-25).

For filamentous fungal host cells, suitable promoters include, but arenot limited to, promoters obtained from the genes for Aspergillus oryzaeTAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucormiehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphate isomerase, Aspergillus nidulans acetamidase, andFusarium oxysporum trypsin-like protease (see, e.g., WO 96/00787, whichis hereby incorporated by reference herein), as well as the NA2-tpipromoter (a hybrid of the promoters from the genes for Aspergillus nigerneutral alpha-amylase and Aspergillus oryzae triose phosphateisomerase), and mutant, truncated, and hybrid promoters thereof.

In a yeast host, useful promoters can be from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GALI), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP), andSaccharomyces cerevisiae 3-phosphoglycerate kinase. Other usefulpromoters for yeast host cells are described by Romanos et al., 1992,Yeast 8:423-488.

The control sequence may also be a suitable transcription terminatorsequence, that is, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleic acid sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used.

For example, exemplary transcription terminators for filamentous fungalhost cells can be obtained from the genes for Aspergillus oryzae TAKAamylase, Aspergillus niger glucoamylase, Aspergillus nidulansanthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusariumoxysporum trypsin-like protease.

Exemplary terminators for yeast host cells can be obtained from thegenes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYCI), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA that is important for translation by thehost cell. The leader sequence is operably linked to the 5′ terminus ofthe nucleic acid sequence encoding the polypeptide. Any leader sequencethat is functional in the host cell of choice may be used. Exemplaryleaders for filamentous fungal host cells are obtained from the genesfor Aspergillus oryzae TAKA amylase and Aspergillus nidulans triosephosphate isomerase. Suitable leaders for yeast host cells are obtainedfrom the genes for Saccharomyces cerevisiae enolase (ENO-1),Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomycescerevisiae alpha-factor, and Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleic acid sequence andwhich, when transcribed, is recognized by the host cell as a signal toadd polyadenosine residues to transcribed mRNA. Any polyadenylationsequence which is functional in the host cell of choice may be used inthe present invention. Exemplary polyadenylation sequences forfilamentous fungal host cells can be from the genes for Aspergillusoryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillusnidulans anthranilate synthase, Fusarium oxysporum trypsin-likeprotease, and Aspergillus niger alpha-glucosidase. Usefulpolyadenylation sequences for yeast host cells are described by Guo &Sherman, 1995, Mol. Cell Bio. 15:5983-5990.

The control sequence may also be a signal peptide coding region thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleic acidsequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion that encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region thatis foreign to the coding sequence. The foreign signal peptide codingregion may be required where the coding sequence does not naturallycontain a signal peptide coding region.

Effective signal peptide coding regions for bacterial host cells are thesignal peptide coding regions obtained from the genes for Bacillus NClB11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase,Bacillus licheniformis subtilisin, Bacillus licheniformisbeta-lactamase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen & Palva, 1993, Microbiol. Rev. 57: 109-137.

Effective signal peptide coding regions for filamentous fungal hostcells can be the signal peptide coding regions obtained from the genesfor Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, and Humicola lanuginosa lipase.

Useful signal peptides for yeast host cells can be from the genes forSaccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding regions are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding region that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from thegenes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilisneutral protease (nprT), Saccharomyces cerevisiae alpha-factor,Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophilalactase (see, e.g., WO 95/33836).

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

It may also be desirable to include regulatory sequences that permitregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. In prokaryotic host cells, suitable regulatory sequencesinclude the lac, tac, and trp operator systems. In yeast host cells,suitable regulatory systems include, as examples, the ADH2 system orGALI system. In filamentous fungi, suitable regulatory sequences includethe TAKA alpha-amylase promoter, Aspergillus niger glucoamylasepromoter, and Aspergillus oryzae glucoamylase promoter.

Other examples of regulatory sequences are those that allow for geneamplification. In eukaryotic systems, these include the dihydrofolatereductase gene, which is amplified in the presence of methotrexate, andthe metallothionein genes, which are amplified with heavy metals. Inthese cases, the nucleic acid sequence encoding the transaminasepolypeptide of the present invention would be operably linked with theregulatory sequence.

Thus, in another embodiment, the present disclosure is also directed toa recombinant expression vector comprising a polynucleotide encoding avariant LovD polypeptide, or a catalytically active fragment thereof,and one or more expression regulating regions such as a promoter and aterminator, a replication origin, etc., depending on the type of hostsinto which they are to be introduced. The various nucleic acid andcontrol sequences described above may be joined together to produce anexpression vector which may include one or more convenient restrictionsites to allow for insertion or substitution of the nucleic acidsequence encoding the polypeptide at such sites. Alternatively, thenucleic acid sequence of the present disclosure may be expressed byinserting the nucleic acid sequence or a nucleic acid constructcomprising the sequence into an appropriate vector for expression. Increating the expression vector, the coding sequence positioned withinthe vector so that the coding sequence is operably linked with theappropriate control sequences for expression.

The expression vector may be any vector (e.g., a plasmid or virus),which can be conveniently subjected to recombinant DNA procedures andcan bring about the expression of the polynucleotide sequence. Thechoice of the vector will typically depend on the compatibility of thevector with the host cell into which the vector is to be introduced. Thevectors may be linear or closed circular plasmids.

The expression vector may be an autonomously replicating vector, i.e., avector that exists as an extrachromosomal entity, the replication ofwhich is independent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

The expression vector may contain one or more selectable markers, whichpermit easy selection of transformed cells. A selectable marker is agene the product of which provides for biocide or viral resistance,resistance to heavy metals, prototrophy to auxotrophs, and the like.Examples of bacterial selectable markers are the dal genes from Bacillussubtilis or Bacillus licheniformis, or markers, which confer antibioticresistance such as ampicillin, kanamycin, chloramphenicol, ortetracycline resistance. Suitable markers for yeast host cells are ADE2,HISS, LEU2, LYS2, MET3, TRP1, and URA3.

Selectable markers for use in a filamentous fungal host cell include,but are not limited to, amdS (acetamidase), argB (ornithinecarbamoyltransferase), bar (phosphinothricin acetyltransferase), hph(hygromycin phosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Embodiments for use in an Aspergillus cell include the amdS and pyrGgenes of Aspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

Expression vectors for expressing the variant LovD polypeptides cancontain an element(s) that permits integration of the vector into thehost cell's genome or autonomous replication of the vector in the cellindependent of the genome. For integration into the host cell genome,the vector may rely on the nucleic acid sequence encoding thepolypeptide or any other element of the vector for integration of thevector into the genome by homologous or nonhomologous recombination.

Alternatively, the expression vector may contain additional nucleic acidsequences for directing integration by homologous recombination into thegenome of the host cell. The additional nucleic acid sequences enablethe vector to be integrated into the host cell genome at a preciselocation(s) in the chromosome(s). To increase the likelihood ofintegration at a precise location, the integrational elements shouldpreferably contain a sufficient number of nucleic acids, such as 100 to10,000 base pairs, preferably 400 to 10,000 base pairs, and mostpreferably 800 to 10,000 base pairs, which are highly homologous withthe corresponding target sequence to enhance the probability ofhomologous recombination. The integrational elements may be any sequencethat is homologous with the target sequence in the genome of the hostcell. Furthermore, the integrational elements may be non-encoding orencoding nucleic acid sequences. On the other hand, the vector may beintegrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. Examples of bacterial origins of replication are PISAori (as shown in the plasmid of FIG. 5) or the origins of replication ofplasmids pBR322, pUC19, pACYC177 (which plasmid has the PISA ori), orpACYC184 permitting replication in E. coli, and pUB110, pE194, pTA1060,or pAMβ1 permitting replication in Bacillus. Examples of origins ofreplication for use in a yeast host cell are the 2 micron origin ofreplication, ARS1, ARS4, the combination of ARS1 and CEN3, and thecombination of ARS4 and CEN6. The origin of replication may be onehaving a mutation which makes it's functioning temperature-sensitive inthe host cell (see, e.g., Ehrlich, 1978, Proc Natl Acad Sci. USA75:1433).

More than one copy of a variant LovD-encoding nucleic acid may beinserted into a host cell to increase production of the gene product. Anincrease in the copy number of the nucleic acid sequence can be obtainedby integrating at least one additional copy of the sequence into thehost cell genome or by including an amplifiable selectable marker genewith the nucleic acid sequence where cells containing amplified copiesof the selectable marker gene, and thereby additional copies of thenucleic acid sequence, can be selected for by cultivating the cells inthe presence of the appropriate selectable agent.

Many of the vectors useful for expressing variant LovD polypeptides arecommercially available. Suitable commercial expression vectors includep3×FLAGTMTM expression vectors from Sigma-Aldrich Chemicals, St. LouisMo., which includes a CMV promoter and hGH polyadenylation site forexpression in mammalian host cells and a pBR322 origin of replicationand ampicillin resistance markers for amplification in E. coli. Othersuitable expression vectors are pBluescriptll SK(−) and pBK-CMV, whichare commercially available from Stratagene, LaJolla Calif., and plasmidswhich are derived from pBR322 (Gibco BRL), pUC (Gibco BRL), pREP4, pCEP4(Invitrogen) or pPoly (Lathe et al., 1987, Gene 57:193-201).

5.5. Methods of Making the LovD Variant Polypeptides and Nucleic Acids

Variant LovD polypeptides and polynucleotides encoding such polypeptidescan be prepared using methods commonly used by those skilled in the art.

Variants of specifically disclosed variants can be obtained bysubjecting the polynucleotide encoding the variant to mutagenesis and/ordirected evolution methods. An exemplary directed evolution technique ismutagenesis and/or DNA shuffling as described in Stemmer, 1994, ProcNatl Acad Sci USA 91:10747-10751; WO 95/22625; WO 97/0078; WO 97/35966;WO 98/27230; WO 00/42651; WO 01/75767 and U.S. Pat. No. 6,537,746 (eachof which is hereby incorporated by reference herein).

Other directed evolution procedures that can be used include, amongothers, staggered extension process (StEP), in vitro recombination (Zhaoet al., 1998, Nat. Biotechnol. 16:258-261), mutagenic PCR (Caldwell etal., 1994, PCR Methods Appl. 3:S136-S140), and cassette mutagenesis(Black et al., 1996, Proc Natl Acad Sci USA 93:3525-3529). Mutagenesisand directed evolution techniques useful for obtaining additionalvariants are also described in the following references: Ling et al.,1997, “Approaches to DNA mutagenesis: an overview,” Anal. Biochem.254(2):157-78; Dale et al., 1996, “Oligonucleotide-directed randommutagenesis using the phosphorothioate method,” Methods Mol. Biol.57:369-74; Smith, 1985, “In vitro mutagenesis,” Ann. Rev. Genet.19:423-462; Botstein et al., 1985, “Strategies and applications of invitro mutagenesis,” Science 229:1193-1201; Carter, 1986, “Site-directedmutagenesis,” Biochem. J. 237:1-7; Kramer et al., 1984, “Point MismatchRepair,” Cell 38:879-887; Wells et al., 1985, “Cassette mutagenesis: anefficient method for generation of multiple mutations at defined sites,”Gene 34:315-323; Minshull et al., 1999, “Protein evolution by molecularbreeding,” Curr Opin Chem Biol 3:284-290; Christians et al., 1999,“Directed evolution of thymidine kinase for AZT phosphorylation usingDNA family shuffling,” Nature Biotech 17:259-264; Crameri et al., 1998,“DNA shuffling of a family of genes from diverse species acceleratesdirected evolution,” Nature 391:288-291; Crameri et al., 1997,“Molecular evolution of an arsenate detoxification pathway by DNAshuffling,” Nature Biotech 15:436-438; Zhang et al., 1997, “Directedevolution of an effective fructosidase from a galactosidase by DNAshuffling and screening,” Proc Natl Acad Sci USA 94:45-4-4509; Crameriet al., 1996, “Improved green fluorescent protein by molecular evolutionusing DNA shuffling,” Nature Biotech 14:315-319; Stemmer, 1994, “Rapidevolution of a protein in vitro by DNA shuffling,” Nature 370:389-391;U.S. Pat. No. 6,117,679 (Stemmer, Sep. 12, 2000); U.S. Pat. No.6,376,246 (Crameri et al., Apr. 23, 2002); U.S. Pat. No. 6,586,182(Patten et al., Jul. 1, 2003); U.S. Pat. App. No. 2008/0220990 (Fox,Sep. 11, 2008); and U.S. Pat. App. No. 2009/0312196 (Colbeck et al.,Dec. 17, 2009).

Variant LovD polypeptides can be obtained via recombinant expression inhost cells, as described above. The expressed variant LovD polypeptidecan be recovered from the cells and or the culture medium using any oneor more of the well known techniques for protein purification,including, among others, lysozyme treatment, sonication, filtration,salting-out, ultra-centrifugation, and chromatography. Suitable reagentsfor lysing and the high efficiency extraction of proteins from bacteria,such as E. coli., are commercially available under the trade nameCelLytic BTM from Sigma-Aldrich of St. Louis Mo.

Chromatographic techniques for isolation and/or purification of thevariant LovD polypeptide include, among others, reverse phasechromatography, high performance liquid chromatography, ion exchangechromatography, gel electrophoresis, and affinity chromatography.Conditions for purifying a particular enzyme will depend, in part, onfactors such as net charge, hydrophobicity, hydrophilicity, molecularweight, molecular shape, etc., and will be apparent to those havingskill in the art. In some embodiments, the engineered transaminases canbe expressed as fusion proteins with purification tags, such as His-tagshaving affinity for metals, or antibody tags for binding to antibodies,e.g., myc epitope tag.

In some embodiments, affinity techniques may be used to isolate and/orpurify the variant LovD polypeptides. For affinity chromatographypurification, any antibody which specifically binds the variant LovDpolypeptide may be used. For the production of antibodies, various hostanimals, including but not limited to rabbits, mice, rats, etc., may beimmunized by injection with an engineered polypeptide. The polypeptidemay be attached to a suitable carrier, such as BSA, by means of a sidechain functional group or linkers attached to a side chain functionalgroup. Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, including but not limited toFreund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,dinitrophenol, and potentially useful human adjuvants such as BCG(bacilli Calmette Guerin) and Corynebacterium parvum.

5.6. Host Cells

In another aspect, the present disclosure provides a host cellcomprising a polynucleotide encoding a variant LovD polypeptide, thepolynucleotide being operatively linked to one or more control sequencesfor expression of the variant LovD in the host cell. Host cells for usein expressing the variant LovD polypeptides described herein are wellknown in the art and include but are not limited to, bacterial cells,such as E. coli, Lactobacillus, Streptomyces and Salmonella typhimuriumcells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiaeor Pichia pastoris (ATCC Accession No. 201178) or filamentous fungalcells (e.g., Aspergillus, Trichoderma, Humicola, or Chrysosporium);insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animalcells such as CHO, COS, BHK, 293, and Bowes melanoma cells; and plantcells. Appropriate culture mediums and growth conditions for theabove-described host cells are well known in the art.

Polynucleotides for expression of the variant LovD polypeptide may beintroduced into cells by various methods known in the art. Techniquesinclude, among others, electroporation, biolistic particle bombardment,liposome mediated transfection, calcium chloride transfection, andprotoplast fusion. Various methods for introducing polynucleotides intocells will be apparent to the skilled artisan.

The preparation of expression vectors suitable for expressing variantLovD polypeptides in E. coli host cells is described in the Examplessection. Such expression vectors can be used to express variant LovDpolypeptides in a variety of different strains of E. coli bacterial hostcells. A particularly suitable E. coli host cell is BL21 and W3110bioH-knockout. (See, e.g., Xie, Xinkai, and Yi Tang, 2007, Applied andEnvironmental Microbiology 73(7): 2054-2060; Xie et al., 2006, Chemistry& Biology 13(11): 1161-1169; and Xie et al., Metabolic Engineering 9(4):379-386).

5.7. Uses

The variant LovD polypeptides described herein catalyze transfer of anacyl group from thioester co-substrates to monacolin J and analogues orderivatives thereof to yield therapeutically important statin compounds.A specific embodiment of this reaction, in which monacolin J isconverted to simvastatin, is illustrated in the boxed region of FIG. 1.Owing to their catalytic and other properties, the variant LovDpolypeptides described herein can be used to make large quantities oftherapeutically important statins, such as simvastatin, from monacolin Jand/or its C8 ester precursors, in high yields. When monacolin J is usedas a starting material, simvastatin can be obtained in a single step.Contrast this with the semi-synthetic methods currently utilized toobtain simvastatin (illustrated in FIG. 1 with dashed arrows).

Accordingly, the present disclosure also provides methods of makingsimvastatin utilizing the variant LovD polypeptides described herein.According to the methods, and in reference to FIG. 1, monacolin Jsubstrate (or a salt thereof such as a sodium salt or an ammonium salt)(12) is contacted with a variant LovD polypeptide in the presence of anα-dimethylbutyryl thioester co-substrate (14) under conditions in whichthe variant LovD polypeptide transfers the α-dimethylbutyryl group tothe C8 position of monacolin J to yield simvastatin (16).

The identity of the α-dimethylbutyryl thioester co-substrate is notcritical. The variant LovD polypeptides accept a wide variety ofthioester co-substrates. Suitable α-dimethylbutyryl thioesterco-substrates useful for producing simvastatin include, but are notlimited to, α-dimethylbutyryl-S—N-acetylcysteamine (“DMB-S-NAC”),α-dimethylbutyryl-S-methylthioglycolate (“DMB-S-MTG”),α-dimethylbutyryl-S-methyl mercaptopropionate “DMB-S-MMP”),α-dimethylbutyryl-S-ethyl mercaptoproprionate (“DMB-S-EMP”),α-dimethylbutyryl-S-methyl mercaptobutyrate (“DMB-S-MMB”),α-dimethylbutyryl-S-merceaptopropionic acid (“DMB-S-MPA”), andoptionally substituted S-alkyl or optionally substitutedS-aryl/heteroaryl thioesters. Any of these thioester co-substrates, ormixtures of such thioester co-substrates, can be used in the methodsdescribed herein.

The α-dimethylbutyryl substrate can be prepared from commerciallyavailable starting materials using conventional methods. An exemplaryreaction for preparing DMB-S-MMP is illustrated below:

Briefly, methyl 3-mercaptopropionate (2) is acylated with2,2-dimethylbutanoyl chloride (4) in the presence ofN,N-diisopropylamine (DIEA) to yield DMB-S-MMP (6). Specific exemplaryreaction conditions are provided in Example 7. Other α-dimethylbutyrylthioester co-substrates can be prepared by routine modification of thesemethods.

The method can be carried out with a purified variant LovD polypeptide,or, alternatively the LovD polypeptide can be added to the reactionmixture in the form of a crude cell lysate, or a semi-purified celllysate fraction. Methods for purifying variant LovD polypeptides to alevel of purity suitable for use in large scale reactions are providedin Example 3.

The monacolin J substrate (or a salt thereof) can be added to thereaction mixture in purified form, or alternatively, it can be generatedin situ by hydrolysis of lovastatin. Methods for obtaining lovastatinand/or monacolin J are well-known. For example, lovastatin can beisolated from A. terreus via well-known methods (see, e.g., Endo, A.,1980, The Journal of Antibiotics 33(3): 334-336; Hendrickson et al.,1999, Chemistry & Biology 6(7): 429-439; Kennedy et al., 1999, Science284(5418): 1368-1372; and Manzoni et al., 2002, Applied Microbiology andBiotechnology 58(5): 555-564). It is also available commercially.

Monacolin J (and salts thereof) can be obtained via alkaline hydrolysisof lovastatin, using conventional methods. A specific exemplary reactionis provided in Example 6.

The reaction can be carried out under a variety of different reactionconditions. Typically the reaction is carried out as a slurry containingabout 0.2 to 10 g/L, often 0.25 to 5 g/L, variant LovD polypeptide, fromabout 1 to 250 g/L, often 50 to 150 g/L, monacolin J substrate (or asalt thereof) and from about 1 to 10 equiv, often 1 to 2 equiv,α-dimethylbutyryl thioester co-substrate. The reaction is typicallycarried out in an aqueous buffer (0 to 300 mM) having a pH in the rangeof pH 7.5 to 10.5, often pH 8.5 to 9.5. The identity of the buffer isnot critical. Suitable buffers include, but are not limited to,triethanolamine (TEA), potassium phosphate, or a buffer may not be used.Reaction temperatures are from 20 to 50° C., often 20 to 40° C.

Aqueous co-solvent systems can also be used. Such co-solvents willtypically include from about 1 to 10% of a polar organic co-solvent.Suitable polar organic co-solvents include, but are not limited to,MeCN, DMSO, isopropyl alcohol (IPA), dioxane, THF, acetone, and MeOH.

It has been discovered that the thiol by product (18 in FIG. 1)generated by the reaction may inhibit the variant LovD polypeptides.Accordingly, it may be desirable to include a thiol scavenging agent inthe reaction mixture. Suitable thiol scavenging agents and methods fortheir use are described in application Ser. No. 61/247,242, titled“Improved Lov-D Acyltransferase Mediated Acylation,” filed Sep. 30,2009, and Application No. PCT/US2010/050253, of the same name filedconcurrently herewith under, the disclosures of which are incorporatedherein by reference. A preferred thiol scavenging agent is activatedcharcoal. When used, it can be included in the reaction mixture in anamount ranging from about 2 to 20 g/L. Alternatively, the product may beprecipitated as a salt, for example, as an ammonium or sodium salt.

Reaction conditions suitable for use with the variant LovD polypeptidesdescribed herein are as follows: 1 to 250 g/L, also 25 to 200 g/L, andoften 50 to 150 g/L, monacolin J sodium salt substrate; 1 to 10 equiv,also 1 to 5 equiv, and often 1 to 2 equiv, DMB-S-MMP co-substrate; 0.2to 10 g/L, often 0.25 to 5 g/L, variant LovD polypeptide (prepared asdescribed in Example 3); 2 to 20 g/L activated charcoal (optional); and0 to 300 mM TEA buffer, pH 7.5 to 10.5, also pH 8.0 to 10.0, and oftenpH 8.5 to 9.5.

The reaction is carried out at a temperature in the range of about 20 to50° C., also 20 to 30° C., and often 20 to 40° C., depending upon thethermostability variant LovD polypeptide used, with agitation orstirring for a duration of about 18 to 48 hours. The progress of thereaction can be monitored by analyzing aliquots via HPLC chromatographyas described in the Examples section.

Following the reaction, simvastatin can be isolated from the reactionmixture and converted to pharmaceutically useful salts, such as theammonium salt, using standard procedures.

Briefly, by-product and excess substrate are extracted with MTBE (2×),the aqueous phases combined and the pH adjusted to pH 5.3-5.4 with 5MHCl while maintaining a temperature of approximately 17° C. EtOAc (13vol) is added and the mixture agitated for 10 minutes with a flat-bladeimpeller (345 rpm, 17° C.). The EtOAc washing process is repeated twicemore and the three EtOAc extractions combined. The EtOAc extractions arefiltered through a Celite pad under reduced pressure, and the filtercake washed with EtOAc. The filtrate and washings are combined andconcentrated under reduced pressure to yield simvastatin hydroxy acid.

The hydroxy acid can be converted to the ammonium salt using standardtechniques. Specific exemplary conditions are provided in Example 8.Alternatively, the reaction can be run using the ammonium salt ofmonacolin J, and the simvastatin ammonium salt produced thereby can beisolated directly from the reaction medium by filtration. This processis exemplified in Example 9.

6. EXAMPLES Example 1 Construction of LovD Genes and Expression Vectors

The acyltransferase encoding gene lovD from wild-type Aspergillusterreus (SEQ ID NO:1) was designed for expression in E. coli usingstandard codon optimization (for a recent review of codon optimizationsoftware, see Puigbò et al., July 2007, “OPTIMIZER: A Web Server forOptimizing the usage of DNA Sequences,” Nucleic Acids Res. 2007 35 (WebServer issue):W126-31). Genes were synthesized using oligonucleotidescomposed of 42 nucleotides and cloned into expression vector pCK110900,depicted in FIG. 3 of U.S. Patent Application Publication No.2006/0195947, which is incorporated herein by reference, under thecontrol of a lac promoter. The expression vector also contained a P15aorigin of replication and a chloramphenicol resistance gene. Resultingplasmids were transformed into E. coli W3110 or E. coli BL21 usingstandard methods.

Polynucleotides encoding exemplary embodiments of the variant LovDpolypeptides of the present disclosure disclosed in Table 1, supra, werealso cloned into vector pCK110900 for expression in E. coli W3110 or E.coli BL21.

Example 2 Shake-Flask Procedure for Production of LovD Polypeptides

A single microbial colony of E. coli containing a plasmid encoding avariant LovD polypeptide of interest was inoculated into 50 mL 2×YTbroth (1× strength, 16 g/L pancreatic digest of casein (tryptonepeptone), 10 g/L yeast extract, 5 g/L sodium chloride) containing 30μg/ml chloramphenicol and 1% glucose. Cells were grown overnight (atleast 16 hours) in an incubator at 30° C. with shaking at 250 rpm. Theculture was diluted into 250 ml 2×YT broth containing 30 μg/mlchloramphenicol, in a 1 liter flask to an optical density at 600 nm(OD600) of 0.2 and allowed to grow at 25-30° C. Expression of the lovDgene was induced by addition of isopropyl β D-thiogalactoside (“IPTG”)to a final concentration of 1 mM when the OD600 of the culture was 0.6to 0.8 and incubation was then continued overnight (at least 16 hours).

Cells were harvested by centrifugation (2400 g, 15 min, 4° C.) and thesupernatant discarded. The cell pellet was re-suspended with an equalvolume of cold (4° C.) 50 mM phosphate buffer (pH 8.5) and harvested bycentrifugation as above. The washed cells were re-suspended in twovolumes of the cold phosphate buffer and passed through a French Press(18,000 psi, 4° C.). Cell debris was removed by centrifugation (7700 g,30 min, 4° C.). The clear lysate supernatant was collected and stored at−20° C. Lyophilization of frozen clear lysate provides a dry shake-flaskpowder of crude LovD polypeptide. Alternatively, the cell pellet (beforeor after washing) can be stored at 4° C. or −80° C.

Example 3 Fermentation Procedure for Production of LovD Polypeptides

Bench-scale fermentations were begun at 37° C. in an aerated, agitated15 L fermentor using 6.0 L of growth medium (0.88 g/L ammonium sulfate,0.98 g/L tri-sodium citrate dihydrate; 12.5 g/L dipotassium hydrogenphosphate trihydrate, 6.25 g/L potassium dihydrogen phosphate, 3.33 g/LTastone-154 yeast extract, 10 mg/L biotin, 0.083 g/L ferric ammoniumcitrate, and 8.3 ml/L of a trace element solution containing 2 g/Lcalcium chloride dihydrate, 2.2 g/L zinc sulfate heptahydrate, 0.5 g/Lmanganese sulfate monohydrate, 1 g/L cuprous sulfate pentahydrate, 0.1g/L ammonium molybdate tetrahydrate and 0.02 g/L sodium tetraborate).The fermentor was inoculated with a late exponential culture of E. coliW3110 or E. coli BL21 containing the plasmid encoding the variant lovDgene of interest (grown in a shake flask as described in Example 2) to astarting OD600 of 0.5 to 2.0. The fermentor was agitated at 500-1500 rpmwith air supplied to the fermentation vessel at 2.0-30.0 L/min tomaintain a dissolved oxygen level of at least 55%. The pH of the culturewas maintained at 7.0 by addition of 28% (v/v) aqueous ammoniumhydroxide. Growth of the culture was maintained by addition of a feedsolution (up to 4 L) containing 500 g/L glucose, 12 g/L ammoniumchloride, 10 mg/L biotin and 5 g/L magnesium sulfate heptahydrate. Afteraddition of 1 L feed volume to the fermentor, at a culture OD600 ofapprox. 50, expression of the lovD gene was induced by addition of IPTGto a final concentration of 1 mM and fermentation continued at 30° C.for another 18 hrs. The culture was then chilled to 4-8° C. andmaintained at that temperature until harvest. Cells were collected bycentrifugation (7300 g, 30 min, 4-8° C. Harvested cells were useddirectly in the recovery process described below or frozen at −20° C.until such use.

The cell pellet was re-suspended and pH adjusted to 8.5 in 2 volumes of100 mM triethanolamine (chloride) buffer (pH 8.5), at 4° C. to eachvolume of wet cell paste. The intracellular LovD polypeptide wasreleased from the cells by passing the suspension through a homogenizerfitted with a two-stage homogenizing valve assembly using a pressure of12000 psi. The cell homogenate was cooled to 4° C. immediately afterdisruption, the pH was adjusted to 8.5, and then a solution of 11% (w/v)polyethyleneimine, pH 7.2, was added to the lysate to a finalconcentration of 0.35-0.5% (w/v) and stirred at 600 rpm for 30 minutesat room temperature of 25-30° C. The resulting suspension was clarifiedby centrifugation (7300 g, 60 min, 4-8° C. The clear supernatant wasdecanted, its pH adjusted to 8.5, and concentrated eight to ten-fold at20° C. using a cellulose ultrafiltration membrane (molecular weight cutoff 30 KDa). The final concentrate was dispensed into petri plates orinto shallow containers, frozen at −20° C. and lyophilized for 48 to 72hr, with the temperature ramping from −20 to 15° C., to yield a driedpowder of crude LovD polypeptide. The crude powder was transferred topolythene bags and stored at −20° C.

Example 4 High-Throughput HPLC Method for Determining Conversion ofMonacolin J Sodium Salt to Simvastatin Sodium Salt

The degree of conversion of Monacolin J sodium salt to Simvastatinsodium salt was determined using an Agilent HPLC 1200 equipped with aGemini® C18 column (4.6×50 mm). For the assay, 10 μL samples were elutedwith a 52% (v/v) aqueous solution of acetonitrile containing 0.1%trifluoroacetic acid (TFA) at a flow rate of 1.5 mL/min and atemperature of 30° C. The eluate was monitored at 238 nm. Under theseconditions, the retention times of Monacolin J acid sodium salt,dimethylbutyryl-S-methylmercaptopropionate (DMB-S-MMP) and Simvastatinsodium salt are approximately 0.8. 2.9, and 3.9 min, respectively. Thedegree of conversion of Monacolin J sodium salt to Simvastatin sodiumsalt can be determined using an area-under-the curve analysis.

Example 5 High-Resolution HPLC Method to Determine Conversion ofMonacolin J Sodium Salt to Simvastatin Sodium Salt

5 μL of the reaction mixture was taken and dissolved in 1.0 mL ofMeCN:water (95:5) mixture. The sample was then centrifuged (300 g, 5min., 25° C.) to remove precipitated enzyme and the supernatant wasanalyzed with HPLC. Conversion of Monacolin J sodium salt to Simvastatinhydroxy acid sodium salt were determined using an Agilent HPLC 1200equipped with a Zorbax Eclipse C18 column (150×4.6 mm, 5 μm) withH₂O+0.1% TFA (A) and acetonitrile +0.1% TFA (B) as eluents at a flowrate of 2.0 mL/min at 30° C. and 238 nm. The analysis was run undergradient method with following time and compositions: 0-1 min, 40% B;1-9 min, 90% B; 9-9.5 min, 90% B; 9.5-10.0 min, 40% B; 10.0-10.5 min,40% B. Retention times of the Monacolin J hydroxy acid, Monacolin J,dimethylbutyryl-S-methylmercaptopropionate (DMB-S-MMP), Simvastatinhydroxy acid and Simvastatin were approximately 2.0, 3.2, 5.9, 6.4 and7.7 minutes, respectively.

Example 6 Preparation of Monacolin J from Lovastatin

To lovastatin (30 g, 0.074 mol) in a 3-neck round bottom flask (RBF)fitted with a condenser was added isopropanol (IPA, 250 mL). KOH pellets(33.2 g, 0.593 mol) and water (3 mL, 0.1 vol) were then added to thestirred suspension. The reaction was stirred at 80° C. (internaltemperature) for 7 h. The reaction was then cooled to ˜50° C. and IPAwas removed under reduced pressure (35° C., 50 mbar) until a finalvolume of ˜100 mL (3.3 vol). Water (110 mL, 3.7 vol) was added to theresidue and the solution was cooled to ˜10° C. in an ice-water bath. 6 MHCl (92 mL, 3.0 vol) was added dropwise to the solution whilemaintaining the internal temperature between 12-17° C. The pH of thesolution was adjusted to a final pH between 3 and 4. The mixture wasthen stirred in an ice-bath for 2 h. The obtained solid was filtered offand washed with water (60-90 mL, 2-3 vol) and then heptane (60 mL, 2vol). The filter cake was vacuum dried at 25° C. for 24 h to yield awhite solid (22.4 g, 90% yield) with >99% purity by HPLC analysis.

Example 7 Preparation of DMB-S-MMP

A solution of N,N-diisopropylethylamine (19.9 mL, 120 mmol) and methyl3-mercaptopropanoate (7.21 60 mmol) in isopropyl acetate (i-PrOAc, 100mL) was cooled to an internal temperature of 2° C. To this vigorouslystirred solution, 2,2-dimethylbutanoyl chloride (8.1 g, 60 mmol) wasadded dropwise over 10 min. The resulting suspension was stirred at 25°C. for 2 h. The reaction was monitored by checking the disappearance ofmethyl 3-mercaptopropanoate using thin-layer chromatography (TLC) onsilica plates. Spots were stained with iodine (eluent: 5% EtOAc/heptane;R_(f) of methyl 3-mercaptopropanoate: 0.20). The reaction was quenchedby addition of saturated ammonium chloride (100 mL) followed by i-PrOAc(100 mL) and the resultant mixture stirred until all solid dissolved.The phases were separated and the organic phase was washed successivelywith 1% aqueous hydrochloric acid (100 mL) and then water (2×50 mL). Theorganic phase was then dried over sodium sulfate, filtered, andconcentrated under reduced pressure (45° C. bath, 50 mm Hg) to obtain acrude mixture as a pale yellow liquid. The crude mixture is subjected tocolumn chromatography over silica gel using a heptane to 2%EtOAc:heptane gradient. Fractions comprising the pure product werecombined and concentrated to afford 10.5 g (80%) of methyl3-(2,2-dimethylbutanoylthio)propionate (DMB-S-MMP).

Example 8 Conversion of Monacolin J Sodium Salt to Simvastatin SodiumSalt, Purification, Isolation of Simvastatin Hydroxy Acid and Conversionof Simvastatin Hydroxy Acid to Simvastatin Hydroxy Acid Ammonium Salt

Variant acyltransferase enzymes, prepared as described in Example 3,were assayed for use in a preparative scale conversion of Monacolin Jsodium salt to Simvastatin sodium salt as follows. A 250 mL 3-neck roundbottom flask (RBF) was equipped with an overhead stirrer, a flat-bladeimpeller and an internal thermometer. The reaction vessel was chargedwith Monacolin J hydroxy acid (5 g, 14.79 mmol). 1M NaOH solution (16.3mL) and de-ionised water (8.6 mL) were added subsequently. The mixturewas stirred until all solid dissolved prior to the addition of thebuffer (˜5 min). TEA buffer solution (33.3 mL, 400 mM, pH=8.5) was addedand the pH of the resultant mixture was adjusted from 9.4 to 9.0 with 5M HCl (0.15 mL) prior to the addition of enzyme. Variant LovD enzyme(0.05 g) was charged to the stirred mixture as a powder. The mixture wasstirred for 5 minutes at 350 rpm at 25° C. to obtain homogeneity.DMB-S-MMP (3.55 mL, 16.27 mmol, 1.1 eq) was added to start the enzymaticreaction. The resulting biphasic mixture was stirred at 350 rpm at 25°C. (internal temperature). HPLC analysis, as described in Example 5, wasperformed on samples taken periodically. Approximately 92% conversionwas obtained after 72 h. When activated charcoal (10 g/L) was addedprior to DMB-S-MMP (to scavenge the by-product, methyl3-mercaptopropanoate), 95-99% conversion could be obtained after 40-48hours. In one embodiment, the variant having the mutations described inVariant No. 116 provides good results according to the above conditions.Reaction conditions, including loading amounts of substrate or enzyme,may need to be optimized for the reactivity profile of other variants.

Simvastatin hydroxy acid sodium salt was purified from the abovereaction as follows. After in-process analysis indicated maximumconversion, the pH of the reaction mixture was adjusted to 9.0 from 8.2using 10 M NaOH solution (0.55 mL). If charcoal was added, the reactionmixture was filtered through a pad of Celite (1.5 g) in a standard G4sintered glass funnel under reduced pressure to remove the charcoal. The250 mL 3-neck RBF was rinsed with deionised water (5 mL), which wasfiltered through the same pad of Celite and then combined with thefiltrate. The filter cake was washed with water (5 mL) and the washingswere collected and combined with the filtrate. MTBE (60 mL; 12 vol) wascharged to the reaction and the mixture agitated at 450 rpm for 10minutes. The 2 phases were then separated and collected separately usinga separatory funnel. The aqueous phase was recharged into the 250 mL3-neck RBF and extracted again with MTBE (30 mL; 6 vol). The phases wereseparated and collected separately.

Conversion of Simvastatin hydroxy acid sodium salt to Simvastatinhydroxy acid was then performed as follows. EtOAc (65 mL; 13 vol) wascharged to the aqueous phase. The pH of the mixture was adjusted to5.3-5.4 using 5 M HCl solution (0.52 mL) and agitated at 450 rpm for 10min at 23-25° C. The phases were allowed to separate in a separatoryfunnel. If an emulsion formed, brine was added to improve separation oftwo phases. The aqueous layer was removed and the EtOAc phase wascollected separately. The aqueous layer was recharged into the 250 ml3-neck RBF and extracted again with EtOAc (65 mL; 13 vol). The biphasicmixture was agitated at 450 rpm for 10 minutes at 23° C. and then thephases were allowed to separate in a separatory funnel and collectedseparately. The separatory funnel was rinsed with EtOAc (5 mL), whichwas then combined with the 1st and 2nd EtOAc extracts. The combinedEtOAc extracts were filtered through a pad of Celite (1 g) in a standardG4 sintered glass funnel under reduced pressure to clarify the extract.The filter cake was washed with EtOAc (10 mL) and the washings werecombined with the filtrate. The filtrate was concentrated from 145 mL to65 mL under reduced pressure.

Conversion of Simvastatin hydroxy acid to Simvastatin hydroxy acidammonium salt was performed as follows. The ethyl acetate solutioncontaining the simvastatin hydroxy acid was charged to a 250 mL 3-neckRBF and the reaction mixture was stirred at 250 rpm at 20-22° C. A 1:1(v/v) mixture of ammonium hydroxide (2.5 mL) and MeOH (2.5 mL) was thenadded dropwise over 10 mins to the reaction mixture, maintaining theinternal temperature at 20-22° C. After complete addition of theammonium hydroxide and MeOH mixture, the resultant mixture was stirredat 260 rpm for 1 h at 20-22° C. The slurry was agitated further for 1 hat 0-5° C. The white solid was then filtered through a standard G4sintered glass funnel under vacuum and the reaction vessel was rinsedwith 6.5 mL of cold EtOAc. The rinse was filtered through the same padof Celite and combined with the filtrate. The filter cake was thenwashed with cold EtOAc (6.5 mL; 1.3 vol). The white solid was dried inthe vacuum oven (2 mm Hg) at 25° C. for 24 h to afford approximately4.3-5.0 g (65-75% isolated yield) of simvastatin hydroxy acid, ammoniumsalt as a white solid with chemical purity 94-97% (AUC, 238 nm).

Example 9 Conversion of Monacolin J Hydroxy Acid Ammonium Salt toSimvastatin Hydroxy Acid Ammonium Salt and Isolation of SimvastatinHydroxy Acid Ammonium Salt

Variant acyltransferase enzymes, prepared as described in Example 3,were assayed for use in a preparative scale conversion of Monacolin Jammonium salt to Simvastatin ammonium salt as follows. A 250 mL 3-neckround bottom flask (RBF) was equipped with an overhead stirrer, aflat-blade impeller and an internal thermometer. The reaction vessel wascharged with Monacolin J hydroxy acid (10 g, 29.58 mmol). Deionizedwater (112.0 mL) and NH₄OH (4.2 mL) were added subsequently. The mixturewas stirred until all solid dissolved prior to the pH adjustment (˜2min). The pH of the resultant mixture was adjusted from 9.2 to 9.0 with5 M HCl (1.5 mL) prior to the addition of enzyme. Variant LovD enzyme(0.10 g) was charged to the stirred mixture as a powder. The mixture wasstirred for 5 minutes at 300 rpm at 25° C. to obtain homogeneity.DMB-S-MMP (7.1 mL, 32.54 mmol, 1.1 eq) was added to start the enzymaticreaction. The resulting biphasic mixture was stirred at 300 rpm at 25°C. (internal temperature). The pH of the reaction was controlled at 9.0by pH stat and titration of 25% NH₄OH. HPLC analysis, as described inExample 5, was performed on samples taken periodically. Approximately97% conversion was obtained after 48 h. In one embodiment, the varianthaving the mutations described in Variant No. 116 provides good resultsaccording to the above conditions. Reaction conditions, includingloading amounts of substrate or enzyme, may need to be optimized for thereactivity profile of other variants.

Simvastatin hydroxy acid ammonium salt was isolated from the abovereaction as follows. After in-process analysis indicated maximumconversion, the reaction mixture was filtered through a standard G4sintered glass funnel under reduced pressure. The 250 mL 3-neck RBF wasrinsed with chilled deionized water (10 mL) and the slurry was filteredthrough the same sintered glass funnel. The filter cake was washed twicewith chilled deionized water (20 mL) and then washed three times withMTBE (40 mL). The white solid was dried in a vacuum oven (2 mmHg) at 25°C. for 24 h to afford approximately 11.4 to 11.7 g (85 to 87% isolatedyield) of Simvastatin hydroxy acid ammonium salt as a white solid with achemical purity of about 97 to 98% (AUC, 238 nm).

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wasindividually indicated to be incorporated by reference for all purposes.

While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the invention(s).

What is claimed is:
 1. An engineered variant LovD polypeptide having atleast two-fold greater catalytic activity than the wild-type Aspergillusterreus acyltransferase of the amino acid sequence of SEQ ID NO: 2,wherein said engineered variant LovD polypeptide has at least 90%sequence identity to the wild-type A. terreus acyltransferase of theamino acid sequence of SEQ ID NO: 2, and wherein said engineered variantLovD polypeptide comprises the substitution G275S.
 2. The engineeredvariant LovD polypeptide of claim 1, wherein said engineered variantLovD polypeptide has at least 10-fold greater catalytic activity thanthe wild-type A. terreus acyltransferase of the amino acid sequence ofSEQ ID NO:
 2. 3. The engineered variant LovD polypeptide of claim 1,wherein said engineered variant LovD polypeptide further comprises oneor more substitutions selected from the group consisting of I4N, A9V,K26E, R28K, R28S, I35L, C40A, C40V, C40F, S41R, N43Y, C60F, C60Y, C60N,C60H, S109C, S142N, A184T, A184V, N191S, A261T, A261E, A261V, L292R,Q297E, L335M, A377V, A383V, N391D, and H404R.
 4. The engineered variantLovD polypeptide of claim 1, further comprising one or more of thefollowing features: (i) at least one substitution selected from thegroup consisting of A123P, M157V, S164G, S172N, L174F, A178L, N191G,L192I, A247S, R250K, S256T, A261H, Q297G, L361M, V370I and N391S; (ii)at least one substitution selected from the group consisting of Q124M,A261H, Q295R AND 0412R; (iii) at least one substitution selected fromthe group consisting of N43R, D96R and H404K; (iv) at least onesubstitution selected from the group consisting of C40R, C60R AND D245E;and (v) at least one substitution selected from the group consisting ofI4N, A9V, K26E, R28K, R28S, I35L, C40A, C40V, C40F, S41R, N43Y, C60F,C60Y, C60N, C60H, S109C, S142N, A184T, A184V, N191S, A261T, A261E,A261V, L292R, Q297E, L335M, A377V, A383V, N391D and H404R.
 5. A methodof making an engineered variant LovD polypeptide comprising culturing ahost cell under conditions in which the engineered variant LovDpolypeptide of claim 1 is expressed and optionally recovering the saidengineered variant LovD polypeptide.
 6. A method of making simvastatincomprising contacting a monacolin J substrate with the engineeredvariant LovD polypeptide of claim 1 in the presence of ana-dimethylbutyryl thioester co-substrate and under conditions in whichthe monacolin J is converted to simvastatin.
 7. The method of claim 6,wherein said monacolin J is a sodium salt.
 8. The method of claim 7,wherein said monacolin J is a sodium salt and activated charcoal isadded.
 9. The method of claim 8, wherein said monacolin J is an ammoniumsalt.
 10. The method of claim 6, wherein an agent for precipitatingsimvastatin is added.